Ethylene Differentially Modulates Hypoxia Responses and Tolerance across Solanum Species

Abstract

The increasing occurrence of floods hinders agricultural crop production and threatens global food security. The majority of vegetable crops are highly sensitive to flooding and it is unclear how these plants use flooding signals to acclimate to impending oxygen deprivation (hypoxia). Previous research has shown that the early flooding signal ethylene augments hypoxia responses and improves survival in Arabidopsis. To unravel how cultivated and wild Solanum species integrate ethylene signaling to control subsequent hypoxia acclimation, we studied the transcript levels of a selection of marker genes, whose upregulation is indicative of ethylene-mediated hypoxia acclimation in Arabidopsis. Our results suggest that ethylene-mediated hypoxia acclimation is conserved in both shoots and roots of the wild Solanum species bittersweet (Solanum dulcamara) and a waterlogging-tolerant potato (Solanum tuberosum) cultivar. However, ethylene did not enhance the transcriptional hypoxia response in roots of a waterlogging-sensitive potato cultivar, suggesting that waterlogging tolerance in potato could depend on ethylene-controlled hypoxia responses in the roots. Finally, we show that ethylene rarely enhances hypoxia-adaptive genes and does not improve hypoxia survival in tomato (Solanum lycopersicum). We conclude that analyzing genes indicative of ethylene-mediated hypoxia acclimation is a promising approach to identifying key signaling cascades that confer flooding tolerance in crops.

Keywords: PRT6 N-degron pathway of proteolysis; Solanum dulcamara; Solanum lycopersicum; Solanum tuberosum; VII Ethylene Response Factor; ethylene; flooding; hypoxia; phytoglobin.

Figure 1

Potato cultivars show variation in waterlogging tolerance. Relative shoot dry weight (a), relative leaf area (b) of 6 elite potato (S. tuberosum) cultivars during 12 days of waterlogging. Values are relative to non-waterlogged plants who were set at 100% per time-point (a,b). Asterisks indicate significant differences between the Festien (purple) and Seresta (pink) cultivars per time-point and these were the cultivars selected for further analysis (indicated by arrows, ** p < 0.01, Generalized linear model, Tukey’s honestly significant difference (HSD), n = 10 plants).

Figure 2

Ethylene differentially modulates hypoxia-responsive transcripts in the roots of elite potato cultivars. Relative mRNA transcript abundance of four marker genes for ethylene-mediated hypoxia tolerance in shoot (a) and root tissues (b) of the potato cultivars Festien (waterlogging tolerant) and Seresta (waterlogging sensitive) after 4 h of pre-treatment with air (light green) or ~5 μL L⁻¹ ethylene (pink), followed by (4 h) hypoxia (green and purple). Marker genes are orthologues of the Arabidopsis genes: ethylene signaling gene ETR2, NO-scavenging phytoglobin PGB1, ERFVII transcription factor RAP2.12 and hypoxia adaptive gene PDC1. Values are relative to air treated samples of Festien. Different letters indicate significant differences (p < 0.05, 2-way ANOVA, Tukey’s HSD, n = 3 containing 2 shoot meristems (a) or ~10 root tips (b)).

Figure 3

Ethylene differentially mediates hypoxia tolerance in Solanum species. Root tip survival of S. dulcamara, S. lycopersicum (Moneymaker), S. melongena and S. pennellii seedlings after 4 h of pre-treatment with air (green) or ~5 μL L⁻¹ ethylene (purple) followed by 4 h (a) or 2 and 2.5 h (b) (only S. pennellii) of hypoxia and 2 days of recovery. Asterisks indicate significant differences between air and ethylene (error bars are SEM, * p < 0.05, Student’s t test, n = 4–10 rows containing 8–10 seedlings). Shoot survival (c,f), phenotypes (d) (only S. dulcamara) and fresh weight (FW) (e,g) of S. dulcamara (c–e) and S. lycopersicum (f,g) plants after 4 h of air (green) or ~5 μL L⁻¹ ethylene (purple) pre-treatment followed by hypoxia and 7 days recovery. Values are relative to control (normoxia) plants. Scale bar = 3 cm. Asterisks indicate significant differences between air and ethylene (error bars are SEM, ** p < 0.01, Student’s t test, n = 10–13 plants).

Figure 4

Ethylene augments hypoxia-responsive transcripts in bittersweet. Relative mRNA transcript abundance of 10 marker genes for ethylene-mediated hypoxia tolerance in bittersweet (S. dulcamara) shoot (a) and root tissues (b) after 4 h of pre-treatment with air (light green) or ~5 μL L⁻¹ ethylene (pink), followed by (4 h) hypoxia (green and purple). Marker genes are orthologues of the Arabidopsis genes: ethylene signaling genes ACO1 and ETR2, NO-scavenging phytoglobins PGB1, PGB2 and PGB3, ERFVII transcription factors RAP2.3 and RAP2.12 and hypoxia adaptive genes SRO5, PDC1 and ADH1. Values are relative to air treated samples. Different letters indicate significant differences (p < 0.05, 1-way ANOVA, Tukey’s HSD, n = three or six biological replicates containing 2–4 shoot meristems (a) or ~10 root tips (b)).

Figure 5

Ethylene rarely enhances hypoxia-responsive transcripts in tomato. Relative mRNA transcript abundance of 10 marker genes for ethylene-mediated hypoxia tolerance in tomato (S. lycopersicum cv MoneyMaker) shoot (a) and root tissues (b) after 4 h of pre-treatment with air (light green) or ~5 μL L⁻¹ ethylene (pink), followed by (4 h) hypoxia (green and purple). Marker genes are orthologues of the Arabidopsis genes: ethylene signaling genes ACO1 and ETR2, NO-scavenging phytoglobins PGB1, PGB2 and PGB3, ERFVII transcription factors RAP2.3 and RAP2.12 and hypoxia adaptive genes SRO5, PDC1 and ADH1. Values are relative to air treated samples. Different letters indicate significant differences (p < 0.05, 1-way ANOVA, Tukey’s HSD, n = three or six biological replicates containing 2–4 shoot meristems (a) or ~10 root tips (b)).

Figure 6

Overview of ethylene-mediated hypoxia responses in Solanum species. (a) Schematic showing the proposed mechanism of ethylene-mediated hypoxia tolerance, as discovered in Arabidopsis [10]. Arrows pointing downward indicate key processes that were verified in Arabidopsis at the mRNA and protein level, or tested in the Solanum species at mRNA level in both roots and shoots. (b) Table showing whether marker genes or hypoxia tolerance (as assessed by regrowth capacity following stress removal) were enhanced by an early ethylene treatment (green check mark = yes, red cross = no, question mark = not tested). The n/n in the hypoxia response column indicates the amount of hypoxia genes that are ethylene-enhanced and hypoxia-responsive upon hypoxia compared to the total amount of hypoxia-responsive genes tested. This table is based on the experimental data shown in Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5 and [10,27].