Genetic Variation for Thermotolerance in Lettuce Seed Germination Is Associated with Temperature-Sensitive Regulation of ETHYLENE RESPONSE FACTOR1 (ERF1)

Abstract

Seeds of most lettuce (Lactuca sativa) cultivars are susceptible to thermoinhibition, or failure to germinate at temperatures above approximately 28°C, creating problems for crop establishment in the field. Identifying genes controlling thermoinhibition would enable the development of cultivars lacking this trait and, therefore, being less sensitive to high temperatures during planting. Seeds of a primitive accession (PI251246) of lettuce exhibited high-temperature germination capacity up to 33°C. Screening a recombinant inbred line population developed from PI215246 and cv Salinas identified a major quantitative trait locus (Htg9.1) from PI251246 associated with the high-temperature germination phenotype. Further genetic analyses discovered a tight linkage of the Htg9.1 phenotype with a specific DNA marker (NM4182) located on a single genomic sequence scaffold. Expression analyses of the 44 genes encoded in this genomic region revealed that only a homolog of Arabidopsis (Arabidopsis thaliana) ETHYLENE RESPONSE FACTOR1 (termed LsERF1) was differentially expressed between PI251246 and cv Salinas seeds imbibed at high temperature (30°C). LsERF1 belongs to a large family of transcription factors associated with the ethylene-signaling pathway. Physiological assays of ethylene synthesis, response, and action in parental and near-isogenic Htg9.1 genotypes strongly implicate LsERF1 as the gene responsible for the Htg9.1 phenotype, consistent with the established role for ethylene in germination thermotolerance of Compositae seeds. Expression analyses of genes associated with the abscisic acid and gibberellin biosynthetic pathways and results of biosynthetic inhibitor and hormone response experiments also support the hypothesis that differential regulation of LsERF1 expression in PI251246 seeds elevates their upper temperature limit for germination through interactions among pathways regulated by these hormones. Our results support a model in which LsERF1 acts through the promotion of gibberellin biosynthesis to counter the inhibitory effects of abscisic acid and, therefore, promote germination at high temperatures.

Figure 1.

Germination percentages of parental lines PI (white circles) and Sal (black circles) across temperatures ranging from 24°C to 40°C. The germination of Sal seeds was completely thermoinhibited above 29°C, while PI seed germination was thermoinhibited above 33°C. Error bars (se) are shown when they exceed the size of the symbols.

Figure 2.

Colocation of a QTL for the HTG trait (Htg9.1) on chromosome 9. This QTL was detected based on germination percentages at 30°C and 35°C of seeds produced in three different environments, as represented by solid, dashed, and dotted lines, and explained 56% of the phenotypic variation observed in the mapping population. The location and year of seed production are indicated in the key in parentheses. LsERF1, a gene encoding an ethylene transcription factor, is located on scaffold 243 that maps to the center of this QTL interval based on allelic assignment of marker NM4182 in F2 and NIL populations. Horizontal thick bars represent the 1 − LOD (log of the odds) QTL confidence intervals. The graphed log of the odds values represent the average permutated threshold (1,000 permutations) for declaring a significant QTL.

Figure 3.

Normalized transcript counts of the LsERF1 gene for PI (circles) and Sal (triangles) based on RNAseq data from lettuce seeds imbibed at 20°C (white symbols) and 30°C (black symbols). PI seeds at both temperatures and Sal seeds at 20°C completed visible germination (radicle emergence) between 12 and 24 h, while Sal seeds at 30°C did not germinate. Error bars (se) are shown when they exceed the size of the symbols (n = 3).

Figure 4.

A, Germination of PI, NIL-PI, Sal, and NIL-Sal seeds imbibed with deionized water in the absence and presence of 10 μL L⁻¹ ethylene. B, Germination of PI, NIL-PI, Sal, and NIL-Sal seeds imbibed with 10 mm AVG in the presence of different ethylene concentrations (0.01, 0.1, 1, and 10 μL L⁻¹). All seeds were pretreated with 10 mm AVG for 24 h prior to ethylene exposure for 72 h at each genotype’s corresponding germination temperature. C, Germination of PI, NIL-PI, Sal, and NIL-Sal seeds in response to exogenous 10 μL L⁻¹ ethylene in the presence of 10 µm ABA. All genotypes were treated with 10 µm ABA for 24 h prior to 10 μL L⁻¹ ethylene exposure for 72 h at each genotype’s corresponding temperature. D, Germination of PI and NIL-PI seeds and Sal and NIL-Sal seeds in the presence of 30 or 100 µm FLU for 72 h. E, Germination of PI, NIL-PI, Sal, and NIL-Sal seeds imbibed with water, 10 mm AVG, and/or 100 µm FLU. Seeds of all genotypes were treated with 10 mm AVG for 24 h prior to adding 100 µm FLU for 72 h to examine the effect of AVG and FLU presence (absence of ethylene and ABA biosynthesis) in recovering the germination capacity of the genotypes. F, Germination of PI, NIL-PI, Sal, and NIL-Sal seeds imbibed with water or 100 µm PAC. Seeds exposed to ethylene were pretreated with 100 µm PAC for 24 h prior to 10 μL L⁻¹ ethylene exposure for 72 h at each genotype’s corresponding threshold germination temperature. These seeds were then treated with 100 µm GA₄ for another 48 h at the same temperatures in the absence of 10 μL L⁻¹ ethylene. A and D show germination assays of PI and NIL-PI seeds performed at 35°C, while assays of Sal and NIL-Sal seeds were performed at 31°C. These temperatures are 3°C above the maximum parental germination temperature thresholds, which are 28°C for Sal and 32°C for PI. In B and E, seeds of PI and NIL-PI were imbibed at 32°C, and Sal and NIL-Sal seeds were imbibed at 29°C. In C and F, seeds of PI and NIL-PI were imbibed at 32°C, and Sal and NIL-Sal seeds were imbibed at 28°C. Error bars indicate se (n = 4).

Figure 5.

Expression of genes associated with the putative candidate gene of Htg9.1 (LsERF1) and ethylene biosynthesis and signaling pathways in seeds of PI at 32°C (A–D), NIL-PI at 32°C (E–H), and Sal at 29°C (I–L) in various conditions (bars from left): water after 6 h of imbibition; water after 12 h of imbibition; 10 mm AVG after 6 h of imbibition; 10 mm AVG after 12 h of imbibition; 10 mm AVG after 24 h of imbibition; or 24 h in 10 mm AVG before being exposed to 1 μL L⁻¹ ethylene for 6 or 12 h. Results are shown for (from left) LsACO1, LsACS1, LsERF1, and LsMAN1. Error bars indicate se (n = 3 biological replicates).

Figure 6.

Expression of genes associated with germination and dormancy in seeds of PI at 32°C (A–D), NIL-PI at 32°C (E–H), and Sal at 29°C (I–L) in various conditions (bars from left): water after 6 h of imbibition; water after 12 h of imbibition; 10 mm AVG after 6 h of imbibition; 10 mm AVG after 12 h of imbibition; 10 mm AVG after 24 h of imbibition; or 24 h of 10 mm AVG before being exposed to 1 μL L⁻¹ ethylene for 6 or 12 h. Results are shown for (from left) LsGA3ox1, LsGA2ox2, LsNCED4, and LsERF104. Error bars indicate se (n = 3 biological replicates).

Figure 7.

Schematic diagram of how the ERF1 transcription factor might act through the GA biosynthetic pathway to promote germination in PI and NIL-PI seeds during high-temperature imbibition, as denoted by the dashed arrow. The LsERF1 transcription factor may activate the transcription of LsGA3ox1 to elevate the biosynthesis of active GA and alter the hormonal ratio of GA and ABA. The presence of GA subsequently promotes the expression of CWRE-associated genes such as LsMAN1 to induce endosperm weakening for radicle protrusion. Not all biochemical steps are shown. Solid lines indicate interactions that have been demonstrated in prior studies. ABA8′ox, ABA-8′-hydroxylase; KO, ent-kaurene oxidase; PDS, phytoene desaturase; SAM, S-adenosyl-Met.