**4. Discussion**

Optimal potato tuber growth occurs around 20 ◦C and plants are susceptible to losses in productivity under hot, arid conditions. Such conditions are expected to increase in the coming decade, therefore functional indicators of plant stress and the cascading effects on the developing tubers were evaluated. In this study, the Canadian potato cultivar "Vigor" was gradually exposed to increasing water deficit to a level of 35% FC beginning at the start of the tuber bulking phase (Figure 1). As seen previously in the literature, fluorescent measurements were not distinguishable between treatments [15] and thus were not dependable indicators of drought stress in potato plants (Table 1). At the plot level, there were no significant differences in yield or tuber number between the treatments (Table 1), however, discrepancies could become more prominent in commercial field production. Canopy temperature was considerably elevated in the drought treatments and has evidence supporting its use for drought stress assessments [30]. Average concentrations of ABA trended upwards in potato tubers exposed to water deficit compared to well-watered controls, suggesting drought responses had been initiated. Gene expression data further corroborated that drought signaling pathways had been activated as there was marked down-regulation of an ABA receptor PYR1, down-regulation of a series of small auxin up-regulated RNA (SAUR) genes involved in cell expansion and organ elongation in response to the environment [31] and the up-regulation of heat shock factor proteins [32] (Table 2). Notably, outside of common changes to the regulation of heat shock proteins, several genes previously identified as differentially expressed in severely drought-stressed potato leaves [32] were inversely regulated in the mildly stressed potato tubers collected in this study. These include the WRKY transcription factor (PGSC0003DMG400001434) and the developmental gene UPA16 (PGSC0003DMG400031742) [32] which had 2-fold and 78-fold increases in expression compared to well-watered control tubers. Lists of genes implicated in drought stress response in potato leaf tissue [33] and potato stolons [34] have been compiled and here we provide those in developing tubers (Table 2; Supplementary Table S1).

Metabolic effects of drought on parameters such as free amino acids, soluble protein, and phenolics were assessed. The gradual drought stress to which the potato tubers were exposed resulted in no significant differences in total free amino acid concentrations, although treatment averages appeared to be divergent with 154.1 and 207.2 μmol g<sup>−</sup><sup>1</sup> in the control and drought treatments, respectively. These differences could be associated with the up-regulation of genes involved in proteolysis (Table 2). Elevated concentrations of proline have been shown to indicate stress in potato leaves [35], however, similar concentrations were observed in tubers irrespective of treatment. The largest changes occurred in the amino acid profile, where concentrations of branched-chain amino acids leucine and isoleucine increased (Figure 2). This indicated a greater proportion of dietary essential amino acids. Increases in branched-chain amino acids are likely attributable to the up-regulation of acetolactate synthase (Table 2), which is the first enzyme in the branched-chain amino acid synthesis pathway [36].

A major fraction (up to 40%) of the soluble protein in potato tubers consists of glycoproteins, known as patatins, that act both as storage proteins and show activity as non-specific lipid acyl hydrolases (LAH) with potential roles in plant defense against biotic stressors [37,38]. In the case of abiotic stress, it was observed that five patatin genes were down-regulated by at least 16-fold with the regulation of gene expression likely occurring via an increased presence of small RNA (Table 3). A possible consequence is reduced protein content in the resulting potato tubers.

As one of the major sources of plant phenolics in the human diet, potatoes have been targeted in breeding for greater total phenolics and antioxidant capacity [3,39]. Phenolic content is known to show a high degree of environmental plasticity with cooler temperatures during the growing period and storage, attributed to higher average accumulation [4,5]. Under drought conditions, expressions of key enzymes required for the biosynthesis of anthocyanins (DFR), flavonoids (FLS) and chlorogenic acid (HCT) were drastically reduced (Figure 4A). Initial flow into the phenylpropanoid pathway through PAL was also reduced, leading to the accumulation of phenylalanine observed in Figure 2. Similar results have been previously observed in the literature [5]. Key enzymes of the carotenoid pathway were also downregulated (Figure 4B). Environmental conditions leading to the repression of phenolic biosynthesis could minimize gains achieved in breeding programs. Regulation of the phenylpropanoid pathway can occur via MYB transcription factors [8], fifteen of which were significantly suppressed under drought (Supplementary Table S1). Unlike previous findings in potato leaves [10], expressions of small RNA and their target MYB transcription factors were not correlated in the drought-stressed tubers. There was therefore no evidence to sugges<sup>t</sup> that small RNA played a role in regulating the phenylpropanoid or carotenoid pathways under drought conditions in potato tubers (Supplementary Table S4).
