Expression of 9-cis-EPOXYCAROTENOID DIOXYGENASE4 is essential for thermoinhibition of lettuce seed germination but not for seed development or stress tolerance

Abstract

Thermoinhibition, or failure of seeds to germinate at warm temperatures, is common in lettuce (Lactuca sativa) cultivars. Using a recombinant inbred line population developed from a lettuce cultivar (Salinas) and thermotolerant Lactuca serriola accession UC96US23 (UC), we previously mapped a quantitative trait locus associated with thermoinhibition of germination to a genomic region containing a gene encoding a key regulated enzyme in abscisic acid (ABA) biosynthesis, 9-cis-EPOXYCAROTENOID DIOXYGENASE4 (NCED4). NCED4 from either Salinas or UC complements seeds of the Arabidopsis thaliana nced6-1 nced9-1 double mutant by restoring germination thermosensitivity, indicating that both NCED4 genes encode functional proteins. Transgenic expression of Salinas NCED4 in UC seeds resulted in thermoinhibition, whereas silencing of NCED4 in Salinas seeds led to loss of thermoinhibition. Mutations in NCED4 also alleviated thermoinhibition. NCED4 expression was elevated during late seed development but was not required for seed maturation. Heat but not water stress elevated NCED4 expression in leaves, while NCED2 and NCED3 exhibited the opposite responses. Silencing of NCED4 altered the expression of genes involved in ABA, gibberellin, and ethylene biosynthesis and signaling pathways. Together, these data demonstrate that NCED4 expression is required for thermoinhibition of lettuce seeds and that it may play additional roles in plant responses to elevated temperature.

Figure 1.

Both Sal and UC Alleles of NCED4 Rescue the Arabidopsis nced6-1 nced9-1 Double Mutant. (A) Germination at 34°C and 22°C of F4 seeds from Arabidopsis nced6-1 nced9-1 double mutants transformed with 35S:Sal-NCED4 or 35S:UC-NCED4. Data for two independent lines for Sal (35S:Sal-130 and 136) and for UC (35S:UC-117 and 119) are shown, along with nced6-1 nced9-1 seeds containing empty vector (CTL), nced6-1 and nced9-1 single mutants, and Columbia wild type (Col). Final germination percentages (+se) are shown after 3 d incubation in the light at 22 or 34°C. (B) Germination of F4 seeds from Arabidopsis nced6-1 nced9-1 double mutant transformed with 35S:Sal-NCED4 or 35S:UC-NCED4 in the presence of 30 μM PAC. Two independent lines for Sal (35S:Sal-130 and 136) and for UC (35S:UC-117 and 119) are shown along with the empty vector control (CTL) and the wild type (Col). Seeds were pretreated with PAC for 3 d at 4°C and subsequently incubated in the light at 20°C for 3 d. Error bars denote se (n ≥ 3).

Figure 2.

Gain- or Loss-of-Function Analysis of Sal-NCED4. (A) Germination of homozygous transformed UC seeds containing ProSal:Sal-NCED4 (UC+Sal-NCED4), of homozygous Sal seeds in which NCED4 is silenced (Sal-RNAi8-4), of UC control seeds (UC-CTL) and of Sal control seeds (Sal-CTL). Seeds were imbibed on 1% agarose in the light at 20°C (left) and 35°C (right) for 36 or 48 h, respectively. (B) Time courses of seed germination of TILLING mutants MIS1, MIS2, STOP1 and the wild type DS at 35°C in the light. Error bars denote se (n = 3). (C) Seed germination of two TILLING mutants (MIS1 and STOP1), two independent RNAi lines (RNAi8-4 and RNAi8-7) and UC after 120 h imbibition at the indicated temperatures in the light. Error bars denote se (n = 3). [See online article for color version of this figure.]

Figure 3.

Silencing of NCED4 Influences the Expression of Seed Dormancy-Related Genes. Normalized mRNA abundances in homozygous RNAi (RNAi8-4) and control (RNAi8-CTL) seeds imbibed at 35°C in the light for genes associated with ABA biosynthesis and signal transduction ([A] to [F]), GA biosynthesis and signal transduction ([G] to [I]), ethylene biosynthesis ([J] and [K]), seed desiccation tolerance (L), temperature perception (M), chromatin remodeling (N), and dormancy maintenance (O). Error bars denote se of three biological replicates.

Figure 4.

Transcriptional Analyses in Developing Seeds of Sal, UC, and a Sal-RNAi-NCED4 Line. Normalized mRNA abundances of: NCED1-4 ([A] to [D]), GA3ox1 (E), GA20ox1 (F), ABI3 (G), and DOG1 (H) in developing seeds of Sal, UC, and a homozygous RNAi-NCED4 line (RNAi8-4). Error bars denote se of three biological replicates.

Figure 5.

High NCED4 Expression Is Associated with Thermoinhibition. (A) and (B) Seed germination of thermosensitive (Sal, DS, W46, and W55) and thermotolerant (UC, W48, and W51) varieties and accessions at 20 and 35°C in the light (A) or in the dark (B) for 3 d. Closed bars, 20°C; open bars, 35°C. (C) and (D) Normalized NCED4 mRNA abundance in dry (0 h) and imbibed seeds at 20 or 35°C, respectively. Error bars denote se (n = 3). (E) Alignment of NCED4 promoters from lettuce genotypes. Boxed sequences are predicted cis-elements based on the PLACE database. NB, no binding site; see the text for descriptions of MYB1AT, ARR1AT, and DOF. The seed germination percentages (Germ) of the genotypes at 35°C are shown at left; W51, W48, and UC are thermotolerant accessions. [See online article for color version of this figure.]

Figure 6.

NCED1-4 Responses to Dehydration Stress. (A) to (D) NCED1-4 expression in response to dehydration. Eight-week-old leaf tissues were allowed to lose 15% of their initial fresh weight at room temperature (Dehydration). In parallel, leaf tissues were incubated in sealed plastic bags containing wet paper towels as controls (CTL). Error bars denote se (n = 3). (E) to (G) NCED2-4 expression in leaves of RNAi-NCED4 lines (RNAi8-2 and RNAi8-7) and controls (CTL) in response to dehydration. Dehydration treatment was as described for (A) to (D) except that 6-week-old leaves were used. Error bars denote se (n = 3).

Figure 7.

NCED1-4 Responses to Heat Stress. Relative mRNA abundances of NCED1-4 in leaves of Sal ([A] to [D]) and UC ([E] to [H]) under heat stress. Leaves were sealed in plastic bags containing wet paper towels and were held at 25 or 40°C for 60 (black bars) or 90 (gray bars) min or were held at 40°C for 60 min followed by 25°C for 30 min (white bars). Results are representative of three independent experiments. Error bars denote se (n = 3) of biological replicates within an experiment.