DOG1 Influences Seed Thermoinhibition in Lettuce.

To explore whether lettuce homologs of AtDOG1 are involved in regulation of seed thermoinhibition, cDNAs with homology to DOG1 were isolated from four genotypes of lettuce having differing germination thermotolerance. Seeds of cv. Salinas (L. sativa; termed Sal) and PI261653 (Lactuca saligna; termed Saligna) were unable to germinate well at 32 °C, whereas seeds of two thermotolerant genotypes, accession US96UC23 (L. serriola) and PI251246 (L. sativa; termed PI), germinated fully at this temperature (SI Appendix, Fig. S1A). Sequences of LsDOG1 cDNAs from the four genotypes were highly homologous and shared ∼50% overall protein amino acid sequence similarity with Arabidopsis DOG1 (54.7% similarity within DOG1 domain regions; SI Appendix, Fig. S2 and Table S1). LsDOG1 is highly expressed in mature lettuce seeds, whereas its transcript level is much lower in leaves and roots (SI Appendix, Fig. S1C). Its expression in seeds decreased as seed maturation temperature increased (SI Appendix, Fig. S1D), as is also the case for AtDOG1 expression in Arabidopsis seeds (17, 18).

Suppression of LsDOG1 expression in Sal lettuce through RNAi caused 56% and 73% reductions in transcript abundance in two independent lines (Fig. 1A). Seeds of these DOG1-RNAi lines were able to germinate fully at 32 °C, whereas seeds of the segregated nontransgenic control line (i.e., Sal) were completely inhibited (Fig. 1 B and C). In contrast, ectopic expression of LsDOG1 under the native Sal DOG1 promoter (P_DOG1:LsDOG1) in the thermotolerant PI lettuce line resulted in increased DOG1 transcripts in mature dry seeds and enhanced seed thermoinhibition at 30 °C (Fig. 1 D–F). A functional homolog of DOG1 is therefore present in lettuce and is involved in regulating germination thermoinhibition.

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Fig. 1.

Regulation by LsDOG1 of lettuce seed thermoinhibition. (A) Relative LsDOG1 mRNA levels in dry seeds of Sal lettuce and its LsDOG1-RNAi lines, 21–5G and 21–1I. (B and C) Seed germination of Sal, 21–5G, and 21–1I lines at 20 and 32 °C. (D) Relative LsDOG1 mRNA levels in dry seeds of PI lettuce and its LsDOG1 over-expression lines, 33–10 and 33–04. (E and F) Seed germination of PI, 33–10, and 33–04 lines at 20 and 30 °C. In D and F, seeds at 20 °C were imbibed for 16 h, whereas seeds at 32 and 30 °C were imbibed for 30 h. Error bars represent SE (n = 3).

DOG1 Influences Seed Thermoinhibition in Arabidopsis.

Functional DOG1 action in Arabidopsis promotes seed dormancy and extends after-ripening periods required for dormancy alleviation (18, 19), whereas fresh seeds of loss-of-function dog1 mutants can fully germinate without after-ripening or cold stratification (17, 19). To test whether DOG1 might be involved in regulating germination thermoinhibition in Arabidopsis, we imbibed seeds of the Columbia (Col) dog1-3 mutant (Salk_000867) at 32 °C. Mutant seeds germinated more than 80% whereas WT seeds germinated less than 20% at that temperature (SI Appendix, Fig. S1B). Molecular complementation showed that coding regions of LsDOG1 genes from thermosensitive and thermotolerant lettuce genotypes were able to rescue the thermoinhibition phenotype when expressed in Arabidopsis dog1-3 plants (SI Appendix, Fig. S1B), indicating that these homologs are all functional and that their role in regulation of seed thermoinhibition is conserved. The presence of functional DOG1 alleles in US96UC23 and PI251246 is consistent with lack of association of this locus with quantitative trait loci for high-temperature germination in these genotypes (27, 34).

Previous work in lettuce and Arabidopsis showed that NCED genes encoding a key enzyme in the ABA biosynthetic pathway are involved in thermoinhibition of germination (24, 28). We tested the interactive effects of DOG1 and ABA on seed thermoinhibition by overexpressing LsDOG1 in an atnced6-1 atnced9-1 double mutant that displays strong germination thermotolerance as a result of reduced ABA biosynthesis (24, 28). Overexpression of LsDOG1 restored a thermosensitive phenotype to atnced6-1atnced9-1 seeds at 32 °C (SI Appendix, Fig. S1E), indicating that DOG1 and ABA can induce thermoinhibition via parallel pathways. We also generated a dog1-3 × nced9-1 double mutant to further examine the interactive effect of both genes on thermoinhibition. At a higher temperature (35 °C), seeds of the single mutants of dog1-3 and nced9-1 and of the double mutant nced6-1 nced9-1 were fully inhibited from germinating, whereas dog1-3 nced9-1 seeds germinated 95% (SI Appendix, Fig. S1F). This additional effect of dog1-3 in the presence of ABA biosynthesis mutants is in contrast to reports that ABA biosynthesis and functional DOG1 are required for induction of primary dormancy (18, 19). However, consistent with previous reports (17⇓⇓–20), overexpression of LsDOG1 under the CaMV35S promoter in Arabidopsis Col plants resulted in deep seed dormancy that could not be alleviated by the ABA biosynthesis inhibitor fluridone, but could be alleviated by GA (SI Appendix, Fig. S3). Thus, DOG1 and ABA can regulate seed primary dormancy and thermoinhibition, and may act in concert or independently depending on the conditions.

DOG1 Influences Flowering Time in Lettuce Through Effects on miR156.

Suppression of DOG1 expression in Sal lettuce caused early flowering (Fig. 2 A–C), as occurred also in wheat (35). Plants from seeds of seven independent transgenic DOG1-RNAi lines that germinated at 32 °C and from segregated control (nontransgenic) seeds were grown in the greenhouse under relatively noninductive day-length conditions for flowering in fall 2013. Salinas DOG1-RNAi lines flowered in ∼120–150 d, whereas control lines did not flower until >200 d (Fig. 2B). Similar results were observed in two DOG1-RNAi lines that were grown in spring 2014, although these plants all flowered earlier because of lengthening days (Fig. 2C).

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Fig. 2.

Regulation of flowering time in lettuce by DOG1. (A) Two independent early-flowering lettuce lines (DOG1-RNAi) and the control Sal line (CTL) are shown at 120 d after seeding. (B) Times to flowering of F2 plants of seven independent transgenic lines and a segregated control line (CTL). Plants were sown in September 2013 and were genotyped for the presence of the transgene (n ≥ 20 plants per line). (C) Times to flowering of two homozygous DOG1-RNAi lines and a control line that were sown in late February 2014 (n ≥ 20 plants per line). (D) Relative levels of miR156 and miR172 and mRNA levels of SPL3, SPL4, and SPL9 in apical meristems of 6-wk-old control (CTL) and DOG1-RNAi (21-5G, 21–1I) lettuce plants; levels were first normalized by LsUBQ10 and are shown relative to those in control for each gene or miRNA. Error bars represent SE (n = 3).

To assess which flowering regulatory pathways were affected by suppression of LsDOG1 expression, transcript levels of key genes in the different pathways were investigated (SI Appendix, Fig. S4). Consistent with early flowering, transcripts of the floral promoter FT were almost 25-fold higher in young DOG1-RNAi lettuce leaves than in similar leaves of the control line (SI Appendix, Fig. S4). Expression patterns of other flowering-related genes were not consistent with effects on the photoperiod-dependent, GA, vernalization, or autonomous flowering pathways (SI Appendix, Supplementary Text and Figs. S4 and S5). However, transcripts of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) 3, 4, and 9 were increased in leaves of the DOG1-RNAi line compared with its control line (SI Appendix, Fig. S4). Furthermore, the relative mRNA levels of SPL3 and 4 were significantly elevated in apical meristems of DOG1-RNAi lines, although SPL9 expression was not affected (Fig. 2D). SPL gene expression is inhibited by DELLA proteins (SI Appendix, Fig. S5), so a decrease in them could result in increased SPL expression (36). However, transcripts of genes encoding two major DELLA proteins (RGL1 and RGA) were unchanged or elevated in DOG1-RNAi leaves (SI Appendix, Fig. S4), making it unlikely that this is the cause of increased SPL expression. SPL transcripts are targeted for degradation by miR156, a regulator of developmental phase transitions in plants, and SPL proteins can induce expression of FT and other genes promoting flowering (30). SPLs also promote production of miR172, a positive regulator of maturation (juvenile to adult) and floral phase transitions (30). Thus, if this mechanism is operative, we would expect early flowering and high SPL transcript levels to be associated with lower miR156 and higher miR172 levels. Consistent with this, miR156 abundance in apical meristems of DOG1-RNAi lines was only one third of that in the control line, whereas miR172 levels were approximately doubled (Fig. 2D). Thus, the early flowering in DOG1-RNAi lettuce plants may be a result of a reduction in miR156 levels, which would result in increased levels of the floral-promoting SPLs and of miR172 (SI Appendix, Fig. S5).

DOG1 Can Influence Flowering in Arabidopsis.

In contrast to early flowering when LsDOG1 expression was suppressed in lettuce, no effects on flowering times were observed in Arabidopsis dog1-3 mutant plants under long-day or short-day conditions (Table 1 and SI Appendix, Table S2). However, when miR156 levels were elevated by overexpression of the gene encoding it, MIR156 (Col-35S:LsMIR156), flowering times were significantly delayed (Fig. 3A), with a majority of plants beginning to flower more than 50 d after germination (DAG) and having more than 36 rosette leaves; in contrast, flowering of homozygous dog1-3 plants also expressing 35S:LsMIR156 was only slightly delayed compared with Col and dog1-3 plants (Table 1 and SI Appendix, Table S2). Further evaluation of 48 dog1-3–35S:LsMIR156 and 72 Col-35S:LsMIR156 independent transgenic lines showed that 87% of the former had flowered within 50 DAG and 95% had less than 30 leaves at flowering, whereas 72% of the latter required more than 50 d to flower and 83% of flowering lines had more than 30 leaves (Table 1 and SI Appendix, Table S2). Although it is possible that sequence homology between the CaMV35S promoter and the T-DNA insertion in dog1-3 could silence the transgene (37), this result was also reproduced in Landsberg erecta (Ler) and its γ-irradiation–induced dog1-1 mutant (19), although the delay in flowering time caused by 35S:LsMIR156 in Ler was not as strong as in Col (Table 1 and SI Appendix, Table S2). As in Arabidopsis, overexpression of LsMIR156 also greatly delayed flowering in Sal lettuce (SI Appendix, Fig. S6). To test whether this phenomenon is specific to lettuce MIR156, we crossed dog1-3 with a Col-35S:AtMIR156 line that exhibits delayed flowering (38). Effects on flowering were essentially identical to those for LsMIR156 (Fig. 3A, Table 1, and SI Appendix, Table S2). The lack of response of dog1-3 plants to overexpression of LsMIR156 or AtMIR156 was evidently caused by reduced conversion of pri-MIR156 transcripts to miR156, as LsMIR156 (or AtMIR156) transcripts were present at equal or greater levels in dog1-3 meristems (Fig. 3B and SI Appendix, Fig. S7F) or leaves (SI Appendix, Fig. S7A) as in Col tissues also expressing LsMIR156 or AtMIR156. The levels of miR156 in dog1-3–35S:LsMIR156 meristems were only 28% or 13% of those in 25-d-old apical meristems or leaves, respectively, of the Col-35S:AtMIR156 plants (Fig. 3B and SI Appendix, Fig. S7B). This was associated with an increase in miR172 and SPL3, 4, 5, and 9 transcripts in dog1-3–35S:LsMIR156 meristems and leaves (Fig. 3B and SI Appendix, Fig. S7 C–E). Similar differences in miR156 and miR172 levels were also observed in Col and dog1-3 meristems in response to overexpression of AtMIR156 (SI Appendix, Fig. S7F).

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Fig. 3.

Regulation of flowering time in Arabidopsis by DOG1. (A) Seven-week-old Col, dog1-3, dog1-3–35S:LsMIR156 (dog1-Ls156), dog1-3 × Col-35S:AtMIR156 (dog1×At156), Col-35S:LsMIR156 (Col-Ls156), and Col-35S:AtMIR156 (Col-At156) plants grown in long days. (B) Relative levels of miR156 and miR172 and mRNA levels of LsMIR156, SPL3, SPL4, SPL5, and SPL9 in apical meristems of Col and dog1-3 plants expressing LsMIR156 at 25 DAG; levels were first normalized by AtACT2, and values relative to those in the Col-LsMIR156 meristems for each gene or miRNA are shown. Error bars represent SE (n = 3). (C) Col-35S:LsMIR172 and dog1-3–35S:LsMIR172 plants grown for 65 d in short days.

It was recently reported that dog1-5 (SALK_022748) is a gain-of-function mutant in which the level of DOG1 protein is greatly increased and seed dormancy is enhanced compared with Col WT (39), as we have confirmed here (SI Appendix, Fig. S8). In this case, the mutation in DOG1 did not negate the effect of MIR156 overexpression on delaying flowering, but rather somewhat enhanced it; 93% of independent dog1-5-LsMIR156 transgenic lines required more than 50 DAG to flower compared with 72% of Col-LsMIR156 plants, with similar data for numbers of leaves at flowering (Table 1 and SI Appendix, Table S2). Interestingly, as for dog1-3, we did not observe a significant difference in flowering time for dog1-5 plants in the absence of MIR156 overexpression (Table 1 and SI Appendix, Table S1). Thus, in contrast to lettuce, DOG1 regulation of flowering time in Arabidopsis is evident only when MIR156 expression is enhanced.

ABA has also been reported to be involved in floral transitions (40), raising the question of whether the early flowering observed in lettuce DOG1-RNAi plants and dog1-3 plants overexpressing MIR156 could be caused by the alteration of ABA contents, as mutants in ABA biosynthetic genes have similar seed germination phenotypes as dog1-3 mutant seeds (SI Appendix, Fig. S1 B and E). However, the delayed flowering time phenotypes conferred by 35S:AtMIR156 and 35S:LsMIR156 in Col plants also occurred in an nced9-1 mutant background (Table 1 and SI Appendix, Table S2). Thus, although phenotypic effects of MIR156 overexpression on flowering depend upon functional DOG1, expression of NCED9, which is primarily seed-expressed and required for thermoinhibition (28), is not necessary.

The results reported here show that loss of DOG1 function negatively affects processing of MIR156 to miR156 in lettuce and Arabidopsis and elevates miR172 abundance. As miR172 is known to promote early flowering in Arabidopsis (30), we tested the effect of overexpression of LsMIR172 on flowering in Col WT and dog1-3 mutant plants. To maximize phenotypic differences, we grew 100 (for Col-35S:LsMIR172) and 77 (for dog1-3–35S:LsMIR172) independent transgenic lines under short-day conditions. Under short days, WT Col plants developed more than 40 leaves before flowering and 90% of plants did not flower before 90 d, whereas 85% of Col-35S:MIR172 plants had less than 40 leaves at flowering and 39% started flowering in less than 90 DAG (Fig. 3C, Table 1, and SI Appendix, Table S2). This advancement of flowering as a result of overexpression of MIR172 was much reduced in dog1-3 plants: more than 60% of dog1-3–35S:LsMIR172 plants had more than 40 leaves at flowering, as did the dog1-3 plants, and only 9% of dog1-3:35S:LsMIR172 plants flowered in less than 90 DAG (Fig. 3C, Table 1, and SI Appendix, Table S2). Thus, functional DOG1 plays a role in the processing or action of MIR172 as well as of MIR156 (SI Appendix, Fig. S5).

miR156 and miR172 Affect Seed Germination.

Suppression of DOG1 expression in lettuce, which improved germination at warm temperatures (Fig. 1 B and C), also resulted in large reductions in miR156 content in dry seeds, with levels only 5–7% of that in control seeds (Fig. 4A). In mature dog1-3 mutant seeds, the miR156 level was slightly lower than that in mature WT Col seeds, but this difference was more significant when LsMIR156 was overexpressed (Fig.4B). We therefore tested whether expression of MIR156 and MIR172 would affect primary dormancy and thermoinhibition of Arabidopsis seed germination in WT and dog1-3 mutant plants. Expression of 35S:LsMIR156 or 35S:AtMIR156 in Col plants reduced germination at 25 °C, likely reflecting increased primary dormancy, and strongly enhanced seed thermoinhibition at warmer temperatures (Fig. 4C). In contrast, overexpressing LsMIR156 in dog1-3 plants did not affect germination at 25, 30, or 32 °C (Fig. 4C). Although seeds of the nced9-1 mutant, like dog1-3 seeds, were thermotolerant, expression of 35S:AtMIR156 in nced9-1 plants had similar effects in suppressing germination as in Col plants (Fig. 4C). This demonstrates that elevation of miR156 can inhibit germination independently of seed ABA biosynthesis and that the effect of MIR156 expression on seed germination inhibition is dependent on functional DOG1. In contrast, overexpression of LsMIR172 promoted germination of fresh (primary dormant) Col seeds, even at elevated temperatures (Fig. 4D). As dog1-3 seeds did not exhibit primary dormancy and germinated fully at all temperatures tested, an additional effect of LsMIR172 overexpression on germination of dog1-3 seeds could not be detected (Fig. 4D). Thus, miR156 and miR172 can influence seed thermoinhibition as well as flowering and other developmental phase transitions (SI Appendix, Fig. S5).

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Fig. 4.

Lettuce seed content of miR156, effects of modification of miR156 or miR172 expression on germination, and effects of dog1 mutations on expression of DICER-related genes. (A) Levels of miR156 in dry seeds of control and three homozygous DOG1-RNAi lettuce lines, 21–6A, 21–5G, and 21–1I. (B) miR156 levels in dry seeds of Col, dog1-3 mutant, Col expressing LsMIR156, and dog1-3 mutants expressing LsMIR156. (C) Effect of expression of 35S:AtMIR156 and 35S:LsMIR156 on germination of seeds of Col, nced9-1, and dog1-3 genotypes of Arabidopsis. All seeds were tested at 3 wk after harvesting. (D) Germination at 25, 30, or 32 °C of Col WT or dog1-3 mutant seeds and of seeds of these genotypes that had been transformed to express 35S:LsMIR172. Seeds were tested 5 d after harvesting when primary dormancy was still present in Col-WT seeds to demonstrate alleviation of dormancy by LsMIR172 overexpression. (E–G) Transcripts of genes associated with miRNA processing or transcription (DCL1, HYL1, TGH, SE, and CDC5) in dry seeds of Col and its T-DNA dog1-3 (loss-of-function) and dog1-5 (gain-of-function) mutants (E), of Ler-CviDOG1 introgression line and its irradiation-induced mutant dog1-1 (F), and of Salinas lettuce (CTL) and its DOG1-RNAi silencing line (21–1I) (G). mRNA levels were first normalized by AtACT2; values relative to those in Col (E), in Ler-CviDOG1 (F), and in Sal (G) are shown. Error bars represent SE (n = 3).

Altered DOG1 Expression or Function Affects Expression of Genes Encoding miRNA Processing Proteins.

As the conversion of primary MIR156 to miR156 was greatly compromised in seeds of lettuce DOG1-RNAi suppression lines and of Arabidopsis dog1-3 mutants (Fig. 4 A and B), we tested whether transcripts of genes encoding proteins associated with miRNA transcription or processing were altered. After preliminary screening, we focused on five genes: DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES1 (HYL1), the G-patch domain protein TOUGH (TGH), the zinc finger protein SERRATE (SE), and CELL DIVISION CYCLE 5 (CDC5) that binds RNA polymerase II and MIR promoters to positively regulate transcription of MIR-encoding genes (41, 42). Although the specific effects varied somewhat among the Arabidopsis and lettuce genotypes and mutants tested, transcript levels of all of these genes differed from their respective WTs in at least some cases (Fig. 4 E–G). For example, the transcript levels of DCL1, HYL1, and CDC5 in dry dog1-3 mutant seeds were 56%, 55%, and 76%, respectively, of those in dry Col WT seeds, whereas expression of these genes (and of TGH and SE) was elevated compared with Col-WT in seeds of the gain-of-function dog1-5 mutant (Fig. 4E). Consistent reduction in expression of these genes was also observed in dry seeds of the dog1-1 mutant compared with those in Ler-CviDOG1 (Fig. 4F), which contains a strong CviDOG1 allele (19), and transcripts of DCL1, SE, TGH, and CDC5 were also lower in dry DOG1-RNAi lettuce seeds (Fig. 4G). These results indicate that DOG1 could affect miR156 (and possibly miR172) processing in seeds through altering expression of genes encoding miRNA-processing proteins (SI Appendix, Fig. S5).