Effect of wet storage conditions on potato tuber transcriptome, phytohormones and growth

Abstract

Background: Stored potato (Solanum tuberosum L.) tubers are sensitive to wet conditions that can cause rotting in long-term storage. To study the effect of water on the tuber surface during storage, microarray analysis, RNA-Seq profiling, qRT-PCR and phytohormone measurements were performed to study gene expression and hormone content in wet tubers incubated at two temperatures: 4 °C and 15 °C. The growth of the plants was also observed in a greenhouse after the incubation of tubers in wet conditions.

Results: Wet conditions induced a low-oxygen response, suggesting reduced oxygen availability in wet tubers at both temperatures when compared to that in the corresponding dry samples. Wet conditions induced genes coding for heat shock proteins, as well as proteins involved in fermentative energy production and defense against reactive oxygen species (ROS), which are transcripts that have been previously associated with low-oxygen stress in hypoxic or anoxic conditions. Wet treatment also induced senescence-related gene expression and genes involved in cell wall loosening, but downregulated genes encoding protease inhibitors and proteins involved in chloroplast functions and in the biosynthesis of secondary metabolites. Many genes involved in the production of phytohormones and signaling were also affected by wet conditions, suggesting altered regulation of growth by wet conditions. Hormone measurements after incubation showed increased salicylic acid (SA), abscisic acid (ABA) and auxin (IAA) concentrations as well as reduced production of jasmonate 12-oxo-phytodienoic acid (OPDA) in wet tubers. After incubation in wet conditions, the tubers produced fewer stems and more roots compared to controls incubated in dry conditions.

Conclusions: In wet conditions, tubers invest in ROS protection and defense against the abiotic stress caused by reduced oxygen due to excessive water. Changes in ABA, SA and IAA that are antagonistic to jasmonates affect growth and defenses, causing induction of root growth and rendering tubers susceptible to necrotrophic pathogens. Water on the tuber surface may function as a signal for growth, similar to germination of seeds.

Keywords: Defence; Energy metabolism; Growth; Microarray; Oxygen; Potato; RNA-Seq; Storage; Tuber; Water.

Fig. 1

Top Gene Ontology (GO) Classes of up- and downregulated genes in microarray analysis. Expressed genes in potato tubers of cultivar ‘Bintje’ incubated one week in wet or dry conditions at 4 °C were compared. Log2 fold change of ≥ + 1 or ≤ − 1 and FDR value of ≤0.05 were used to select the differentially expressed genes. The y-axis indicates the number of probes in each function class, while the x-axis indicates the GO categories

Fig. 2

RNA-Seq profiling of potato tubers incubated in wet conditions at 15 °C. a Venn diagram of statistically significant differentially expressed genes (DEGs) in potato tubers incubated in wet conditions for 1 h, 24 h and 1 week shows that the responses at 24 h and 1 week are more similar to each other than those at 1 h. b Heat map of all the genes that were identified as statistically significant DEGs in at least one of the samples, based on clustering of 485 DEGs identified with NOIseq with probability > 0.8, p-value < 0.05 and log2 fold change ≥2 and ≤ − 2. Hierarchical clustering suggests that the 24 h and 1 week samples cluster together, while a difference was observed when compared to the 1 h sample. The scale bar at the top represents relative expression values. Red color indicates genes that were upregulated, and green color indicates genes that were downregulated. Black indicates genes whose expression is unchanged in wet tubers compared to dry tubers

Fig. 3

Heat maps with the primary RNA-Seq data of tubers incubated in wet conditions. a Heat map of sugar and amino acid genes show mostly upregulated gene expression. b Heat map of genes involved in phytohormone biosynthesis and signaling show downregulated gene expression linked to jasmonates and upregulated expression linked to other phytohormones. The rows represent statistically significant differentially expressed transcripts, and the columns depict three time-points. The rows are labeled with individual gene function, and the postulated function or hormonal pathway is indicated. The scale bar at the top represents relative expression values

Fig. 4

qRT-PCR validation of differentially expressed genes in wet potato tubers. Correlation coefficient analysis between microarray or RNA-Seq data (x-axis) and qRT-PCR (y-axis) data (log2 fold change) analyzed by the Pearson test (P < 0.05). The trend line equation and the corresponding strong square regression coefficient (R2) are shown. The classification of the genes and the primers used are presented in Additional file 4, and qRT-PCR data on the individual genes are presented in Additional file 5

Fig. 5

Reduction of proteinase inhibitor activity in potato tubers incubated in wet conditions. Three potato tuber cultivars were incubated in wet or dry conditions for one week. To measure the activity of the proteinase inhibitors, the potato extracts were incubated with three commercial proteinases, after which the proteinase activities were measured with the azocasein method. The percentages of protease inhibition were calculated by comparing the activities before and after incubation, with the negative control containing no potato extract, and calculated using the least significant difference test at P < 0.05 (p < 0001). The error bars represent the 95% confidence intervals for the estimated proportions representing four replicates of five tubers per protease assay

Fig. 6

Effect of ethylene inhibitor and methyl jasmonate treatment on gene expression in wet potato tubers. Incubation of potato tubers in wet conditions in the presence of ethylene inhibitor 1-methylcyclopropane (1-MCP) or methyl jasmonate (MeJA) reduced the effect of wet conditions in qRT-PCR analysis. The chosen genes representing different functions were classified into four groups: a Defense responses: CCR4 associated factor 1-like (CAF1), WRKY-type DNA binding protein (WRKY), spermidine synthase 1-like (SPDS), major latex-like protein-like 34 (MLP34), patatin (PAT), defensin protein precursor (PDF); b Hormone biosynthesis and signaling: Nt-iaa4.5 deduced protein (AUX/IAA), lipoxygenase 1 (LOX1), lipoxygenase 3 (LOX3), protein TIFY 10A-like (JAZ1), protein phosphatase 2C 63 (PP2C), harpin inducing protein-like (HIN1); c Sugar and amino acid metabolism: Trehalose-6P synthase (TPS), SNF1-related protein kinase regulatory subunit gamma-1 (KING1), glucose-6-P translocator 2 (GPT2), glycerol kinase (GK), bidirectional sugar transporter (Sweet), alanine aminotransferase 2 (AlaAT2); d Cell wall and secondary metabolism: Expansin (EXP), early nodulin 93 (ENOD), 4-coumarate CoA ligase-like 2 (4CL), HMG-CoA reductase (HMG). The graph demonstrates means of log2 fold change from three independent experiments, error bars show standard error of mean and t-test results are shown (*p < 0.05, **p < 0.01)

Fig. 7

Phytohormone concentrations in wet and dry potato tubers. Concentrations of abscisic acid (ABA), 12-oxo-phytodienoic acid (OPDA), salicylic acid (SA), indole-3-acetic acid (IAA) and jasmonic acid (JA) were measured after incubation of the tubers for one week at 15 °C. The numbers are means of five samples each containing tissue from five tubers. Error bars show standard error and t-test values are shown (**p < 0.01, ***p < 0.001)

Fig. 8

Sprouting and growth of potato tubers after incubation in wet and dry conditions. a Statistical analysis of stem numbers of non-sprouted potato tubers of three cultivars after incubation for one week in wet or dry conditions at 4 °C or 15 °C. Both temperature (p < 0.0001) and incubation in wet conditions (p = 0.003) had a significant effect on the number of stems during growth of the plants in the greenhouse. b Wet incubation of sprouted tubers at 22 °C led to root growth, while the control tubers incubated in dry conditions did not show any root growth

Fig. 9

Model of the main identified signaling pathways involved in the response of potato tubers to wet conditions. The main groups of differentially expressed genes in potato tubers in wet conditions (upregulated, red; downregulated, green) and the postulated hormones affecting their expression are indicated. T6P, trehalose 6-phosphate; ET, ethylene; SA, salicylic acid; JA, jasmonic acid; OPDA, 12-oxo-phytodienoic acid; ABA, abscisic acid; and IAA, auxin are indicated. Solid arrows indicate upregulated effect, blocked arrows suggest negative effect and dashed arrow indicate reduced jasmonate signalling in wet tubers