DELAY OF GERMINATION1 (DOG1) regulates both seed dormancy and flowering time through microRNA pathways

Abstract

Seed germination and flowering, two critical developmental transitions in plant life cycles, are coordinately regulated by genetic and environmental factors to match plant establishment and reproduction to seasonal cues. The DELAY OF GERMINATION1 (DOG1) gene is involved in regulating seed dormancy in response to temperature and has also been associated genetically with pleiotropic flowering phenotypes across diverse Arabidopsis thaliana accessions and locations. Here we show that DOG1 can regulate seed dormancy and flowering times in lettuce (Lactuca sativa, Ls) and Arabidopsis through an influence on levels of microRNAs (miRNAs) miR156 and miR172. In lettuce, suppression of LsDOG1 expression enabled seed germination at high temperature and promoted early flowering in association with reduced miR156 and increased miR172 levels. In Arabidopsis, higher miR156 levels resulting from overexpression of the MIR156 gene enhanced seed dormancy and delayed flowering. These phenotypic effects, as well as conversion of MIR156 transcripts to miR156, were compromised in DOG1 loss-of-function mutant plants, especially in seeds. Overexpression of MIR172 reduced seed dormancy and promoted early flowering in Arabidopsis, and the effect on flowering required functional DOG1 Transcript levels of several genes associated with miRNA processing were consistently lower in dry seeds of Arabidopsis and lettuce when DOG1 was mutated or its expression was reduced; in contrast, transcript levels of these genes were elevated in a DOG1 gain-of-function mutant. Our results reveal a previously unknown linkage between two critical developmental phase transitions in the plant life cycle through a DOG1-miR156-miR172 interaction.

Keywords: DOG1; flowering; lettuce; miRNA; seed dormancy.

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).

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).

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.

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).