1 INTRODUCTION
Environmental stress represents the most important single factor limiting crop yield worldwide, a situation that will certainly aggravate in the near future as a consequence of global climate changes (IPCC 2014; Buchanan, B.B., Gruissem, W. & Jones 2015). Among the adverse conditions that constrain plant growth and reproduction, drought has the highest impact in quantitative terms (FAO 2014). Projections of increased desertification in several Mediterranean-type environments of Europe, Australia, South America and sub-Saharan Africa are consistent across simulation models (IPCC 2014). Water restriction negatively affects photosynthetic rates by decreasing CO2availability as a result of stomatal closure, and by feedback inhibition due to limitations in photosynthate transport to sink organs (Romero, Alarcón, Valbuena & Galeano 2017; Urban, Aarrouf & Bidel 2017). Many changes associated to water deficit are therefore detected in the leaves and accordingly, drought responses are inextricably linked to photosynthesis and chloroplast biochemistry (Zingaretti, Inácio, de Matos Pereira, Paz & de Castro França 2013).
Potato (Solanum tuberosum L.) is the third most important food crop in the world (Visser et al. 2009; FAO 2014), and is vulnerable to drought, salinity and other environmental stresses, which affect tuber yield and quality (Vasquez-Robinet et al. 2008; Muñiz García, Cortelezzi, Fumagalli & Capiati 2018). The situation is particularly critical in developing countries where potato is most important as an affordable and nutritionally rich food supply (Romeroet al. 2017), and where the impact of global climate change is predicted to be more severe (IPCC 2014; Muñiz García et al.2018). Therefore, breeders face increasing pressure to develop new lines with improved drought tolerance while keeping high crop yield, tuber quality and market acceptance (Romero et al. 2017).
Water limitation elicits a very complex plant response, which combines physiological, cellular and metabolic adaptations to the stress situation, and involves genome-wide changes in gene expression patterns. While many drought-responsive genes have been identified, it is presently difficult to define the role played by most of them in the tolerance against this environmental challenge (André et al.2009). As most other abiotic stresses, water deficit causes a rise of reactive oxygen species (ROS) levels, especially in leaves (Gómez, Vicino, Carrillo & Lodeyro 2019). In turn, the drought response of genes involved in ROS metabolism depends on the plant cultivar, its degree of tolerance and the duration and intensity of the stress treatment (Cruz De Carvalho 2008). Vasquez-Robinet et al. (2008) found that the higher drought tolerance displayed by Andean potato genotypes was related to enhanced expression of genes encoding antioxidant proteins located in chloroplasts. Moreover, potato transformation with genes related to ROS scavenging led to lines exhibiting improved performance under water deprivation (Ahmad et al. 2010; Eltayebet al. 2011; Cheng, Deng, Kwak, Chen & Eneji 2013), indicating that manipulation of ROS metabolism is a promising strategy to improve drought tolerance (Gómez et al. 2019).
We have generated tobacco plants with increased tolerance to multiple sources of abiotic stress by introducing a cyanobacterial flavodoxin (Fld) directed to chloroplasts (Tognetti et al. 2006; Zurbriggenet al. 2008). Fld is an electron carrier flavoprotein present in bacteria and some algae that displays essentially the same activities and redox interactions as the iron-sulfur protein ferredoxin (Fd), including functional integration as a final electron acceptor in the photosynthetic electron transport chain (PETC). Fld expression is normally induced in microorganisms under conditions of iron starvation and environmental stress that cause Fd down-regulation, taking over Fd functions as its levels decline, and preventing over-reduction of the PETC and ROS propagation (Zurbriggen et al. 2008; Pierella Karlusich, Lodeyro & Carrillo 2014). The Fld gene is absent from plant genomes (Pierella Karlusich, Ceccoli, Graña, Romero & Carrillo 2015), but introduction of a plastid-targeted Fld improved delivery of reducing equivalents to productive pathways of the chloroplast, which in turn restricted plastid ROS production and increased tolerance to drought and other stresses (Tognetti et al. 2006, 2007; Zurbriggen et al. 2008; Li et al. 2017).
The need for drought-tolerant potato lines prompted us to evaluate the Fld approach in this crop. e describe herein the preparation and characterization of potato plants expressing a plastid-localized Fld, and displaying improved photosynthesis, growth and tuber yield under conditions of water deprivation. To gain further insights into the mechanism(s) of Fld-associated stress tolerance, we generated genome-wide transcript profiles from wild-type (WT) and Fld-expressing potato leaves at a pre-symptomatic stage of water restriction to evaluate early responses to hydric stress, and combined this approach with metabolic profiling of carbohydrates and amino acids. The results provide a detailed snapshot of how chloroplast redox biochemistry affects gene expression metabolism in a major crop during this agronomically relevant abiotic stress.