*3.1. Physiological Response*

A high degree of variability existed within the four control and four treatment plots for the agronomic and physiological traits measured during this study (Table 1). Both crop yield and tuber number per plot were not found to be significantly di fferent from one another, although di fferences in plot averages were observed. Calculated values from spectral measurements, such as CCI and *Fv*/*Fm*, showed no significant di fferences. However, canopy temperatures measured in plots maintained at 35% FC were 3.9 ◦C higher than control plots with soil moisture maintained at 70% FC (Table 1). ABA concentrations in the well-watered control and water-deficit conditions were 43.9 and 55.4 ng gDW−1, respectively. Variation was high among the control plants, resulting in no significant di fferences between treatments.

**Table 1.** Agronomic and physiological traits averaged for drought (35% field capacity) and control (70% field capacity) plots with standard error of the mean in parentheses. Bold numbers indicate significance (*p* < 0.05) between treatments.


### *3.2. Tuber Amino Acid Fluctuations in Response to Soil Moisture Deficit*

Of the eight essential amino acids, lysine, phenylalanine, isoleucine, and leucine were found to be more abundant in drought-stressed tubers. The largest di fferences were observed in the concentrations of leucine, phenylalanine, and isoleucine which increased by 3×, 2×, and 1.9×, respectively (Figure 2A). Quantities of branched-chain amino acids, a group that includes leucine, isoleucine, and valine, were therefore significantly higher under drought treatment. Histidine and valine were the most abundant essential amino acids in developing potato tubers (Figure 2A). The majority of non-essential amino acids had similar concentrations in developing tubers regardless of the treatment. Only glutamic acid showed a marked increase of 5.75 μmol g<sup>−</sup><sup>1</sup> under reduced soil moisture conditions. Concentrations of cysteine, proline, and serine were highest among all amino acids measured (Figure 2B).

**Figure 2.** Concentrations of essential (**A**) and non-essential (**B**) amino acids in potato tubers (*n* = 4) sampled during the tuber bulking phase and the associated SEM (*p* < 0.05) when subjected to 70% and 35% field capacity. \* significance (*p* < 0.05) between treatments.

### *3.3. Di*ff*erential Gene Expression and Regulatory Cascades in Developing Tubers Under Drought Stress*

Tubers subjected to soil moisture deficit showed both di fferential gene and transcript expression, with 75.2% and 68.8% being down-regulated, respectively. One-fifth of genes with di fferential expression were of unknown function. A full summary of the observed changes is listed in Figure 3A with a list of all di fferentially expressed genes provided in Supplementary Table S1. Down-regulated genes include those with functions in ABA, auxin and ethylene signaling as well as in auxin, carotenoid, and phenylpropanoid biosynthesis. Up-regulated genes have roles in amino acid biosynthesis, function as molecular chaperones and are involved in ubiquitin-driven proteolysis. Gene names, functional annotation, and the corresponding *Arabidopsis* homologs used for pathway mapping can be found in Table 2. Focusing on the regulation of the phenylpropanoid pathway, fifteen annotated MYB transcription factors were down-regulated by more than 4-fold under low-soil moisture conditions. Up-regulated transcription factors include two MYB transcription factors and one LIM transcription factor. No other MYB, KNOX or LIM transcription factors were above the cut-o ff values of 5% FDR and a log-fold change greater than two. Genes with key functions in the phenylpropanoid and carotenoid pathways that were down-regulated in potato tubers at 35% FC are highlighted in Figure 4. Raw read counts for gene expression analysis can be found in Supplementary Table S2.


**Figure 3.** Summaries of di fferentially expressed genes ( **A**) and small RNA (**B**) in potato tubers at 35% FC using a threshold of 4-fold di fference in expression and a 5% FDR. Gene homologs in *A. thaliana* were considered if identity was greater than 50%. Functional annotation clustering to KEGG pathways was based on *Arabidopsis* gene IDs using the DAVID Bioinformatics online resource 6.8 (https://david.ncifcrf.gov/).






**Table 3.** List of differentially expressed small RNA clusters in drought-stressed potato tubers that negatively correlate to target transcript expression. Target

**Figure 4.** Diagrams depicting key enzymes in the phenylpropanoid (**A**) and carotenoid (**B**) biosynthetic pathways. Genes that are significantly down-regulated beyond cut-off values of 5% FDR and a log-fold change greater than 2 are written in blue.

The isolated small RNA were grouped into 87,213 clusters with an additional 10,209 unassigned sequences. Of these, 103 clusters and 126 unassigned sequences were differentially expressed. Additional summary statistics are listed in Figure 3B. Differentially expressed small RNA clusters with identified gene targets are listed in Supplementary Table S3. None of the small RNA clusters with target genes listed in Table 2 showed differential expression between the two treatments. Expression of target MYB transcription factor genes was also not correlated to small RNA cluster expression (Supplementary Table S4). Interestingly, the expression of small RNA clusters primarily targeting patatin genes was significantly up-regulated and negatively correlated to target gene expression (*r* = −0.61). These clusters and their targets can be found in Table 3.
