*2.2. Physiological Measurements*

Physiological measurements were taken at the end of the tuber bulking phase (90th day after planting) with readings recorded consistently between 11:30 a.m. and 12:30 p.m. Canopy temperature was assessed with an infra-red thermal imaging camera (FLIR T530, FLIR Systems. Wilsonville, OR, USA), and leaflet chlorophyll content (CCI) was recorded using a chlorophyll content meter (CCM-200- Apogee Instruments, Logan, UT, USA). Quantum yield of dark-adapted leaflets (*Fv*/*Fm*) was measured using the portable fluorometer FluorPen FP 100 (PSI, DRASO Czech Republic). Detachable clips were used to dark-adapt the leaflets for 20 min, and *Fv*/*Fm* was measured on the adaxial surfaces of the top 3rd and 4th leaflet of each sampled plant (three plants per replication).

### *2.3. Amino Acid Profiling and Abscisic Acid Content*

In both high tunnels from each replication, a tuber was collected from the middle of the plot on 13 September 2017 (106 DAP). The tubers were washed in running water, followed by distilled water, cut into cubes while evading the skin and immediately frozen in liquid nitrogen. Samples were stored at −80 ◦C until further use.

Amino acids were extracted from 10 mg of ground freeze-dried tissue following Inaba et al. (1994) [19] with some modifications. Briefly, 1 mL of 80% (*v*/*v*) ethanol solution (40 ◦C) was added to each sample, shaken for 30 min at 40 ◦C and the supernatant was recovered by centrifugation (4000 rpm for 10 min) at 4 ◦C. The pellets were re-extracted under the same conditions with an additional 500 μL of 80% (*v*/*v*) ethanol (40 ◦C). The supernatants were combined and stored at −20 ◦C until further use. Amino acids were derivatized following Waters AccQTag Reagent Kit (Waters, Milford, MA, USA; [20]). Briefly, a 10 μL aliquot of sample was mixed with 70 μL borate buffer and 20 μL AccQFluor reagen<sup>t</sup> which was reconstituted in acetonitrile. AccQFluor reagen<sup>t</sup> was reconstituted as follows: 1 mL of AccQFluor reagen<sup>t</sup> diluent was transferred to a vial containing AccQFluor reagen<sup>t</sup> powder and vortexed for 10 s before heating at 55 ◦C for a maximum of 10 min or until dissolved. The derivatized mixture was transferred to an autosampler vial and incubated at 55 ◦C for 10 min. High-performance liquid chromatography (HPLC) was conducted, as described in Waters AccQTag's chemistry package instruction manual, with samples separated on a Waters amino acid column −3.9 × 150 mm and quantified at an excitation wavelength of 285 nm and an emission wavelength of 320 nm using a 2475 scanning fluorescence detector (Waters, Milford, MA, USA). The column was set at 37 ◦C with a 5 μL injection volume. Waters AccQTag buffer (100 mL AccQTag Buffer concentrate +1000 mL deionized water), acetonitrile, and deionized water were used as mobile phases A, B, and C, respectively.

Abscisic acid content was determined following Yan et al., (2016) [21]. Samples were centrifuged to remove debris, and the pellet was washed twice. The supernatant was evaporated in a SpeedVac, and reconstituted in 1 mL of 1% (*v*/*v*) acetic acid. Abscisic acid (ABA) was purified by solid-phase extraction using Oasis HLB, MCX, and WAX cartridge columns (Waters, Milford, MA, USA). The solvent was removed under vacuum and subjected to LC-ESI-MS/MS analysis (Agilent 6410 TripleQuad LC/MS system). An LC (Agilent 1200 series) equipped with a 50 × 2.1 mm, 1.8-μm Zorbax SB-Phenyl column (Agilent Technologies, Santa Clara, CA, USA) was used with a binary solvent system comprised of 0.01% (*v*/*v*) acetic acid in water (solvent A) and 0.05% (*v*/*v*) acetic acid in acetonitrile (solvent B). Separations were performed using a gradient of increasing acetonitrile content at a flow rate of 0.2 mL min−1. The gradient was increased linearly from 3% B to 50% B over 15 min. The retention time of ABA was 14 min.
