The role of the Arabidopsis FUSCA3 transcription factor during inhibition of seed germination at high temperature

Abstract

Background: Imbibed seeds integrate environmental and endogenous signals to break dormancy and initiate growth under optimal conditions. Seed maturation plays an important role in determining the survival of germinating seeds, for example one of the roles of dormancy is to stagger germination to prevent mass growth under suboptimal conditions. The B3-domain transcription factor FUSCA3 (FUS3) is a master regulator of seed development and an important node in hormonal interaction networks in Arabidopsis thaliana. Its function has been mainly characterized during embryonic development, where FUS3 is highly expressed to promote seed maturation and dormancy by regulating ABA/GA levels.

Results: In this study, we present evidence for a role of FUS3 in delaying seed germination at supraoptimal temperatures that would be lethal for the developing seedlings. During seed imbibition at supraoptimal temperature, the FUS3 promoter is reactivated and induces de novo synthesis of FUS3 mRNA, followed by FUS3 protein accumulation. Genetic analysis shows that FUS3 contributes to the delay of seed germination at high temperature. Unlike WT, seeds overexpressing FUS3 (ML1:FUS3-GFP) during imbibition are hypersensitive to high temperature and do not germinate, however, they can fully germinate after recovery at control temperature reaching 90% seedling survival. ML1:FUS3-GFP hypersensitivity to high temperature can be partly recovered in the presence of fluridone, an inhibitor of ABA biosynthesis, suggesting this hypersensitivity is due in part to higher ABA level in this mutant. Transcriptomic analysis shows that WT seeds imbibed at supraoptimal temperature activate seed-specific genes and ABA biosynthetic and signaling genes, while inhibiting genes that promote germination and growth, such as GA biosynthetic and signaling genes.

Conclusion: In this study, we have uncovered a novel function for the master regulator of seed maturation, FUS3, in delaying germination at supraoptimal temperature. Physiologically, this is important since delaying germination has a protective role at high temperature. Transcriptomic analysis of seeds imbibed at supraoptimal temperature reveal that a complex program is in place, which involves not only the regulation of heat and dehydration response genes to adjust cellular functions, but also the activation of seed-specific programs and the inhibition of germination-promoting programs to delay germination.

Figure 1

Transcriptional regulation of FUS3 by abiotic stresses. (A-D) Relative FUS3 mRNA levels measured by qPCR in mature seeds exposed to abiotic stresses during imbibition under constant light. A) salt, 150 mM NaCl; B) osmotic, 300 mM mannitol; C) low temperature, 12°C and D) high temperature, 32°C. The mean value of three replicates was normalized using ACTIN 7 as the internal control. Results are plotted as the ratio to the lowest detected level. Two independent experiments were conducted with similar results and one representative is shown.

Figure 2

Seed imbibition at supraoptimal temperature activates the FUS3 promoter and induces de novo FUS3 mRNA synthesis and FUS3 protein accumulation. (A) Relative FUS3 mRNA levels measured by qPCR in seeds imbibed at 21°C or 32°C for up to 72 h under constant light. The mean value of three replicates was normalized using ACTIN 7 as the internal control. Results are plotted as the ratio to the lowest detected level. Two independent experiments were conducted with similar results and one representative is shown. (B) Confocal images showing GFP fluorescence in the epidermis of FUS3:GFP embryos of seeds imbibed at 21°C or 32°C. HAI, hours after imbibition. (C, D) Confocal images (C) and immunoblots (D) showing FUS3-GFP fluorescence and protein accumulation in FUS3p:FUS3-GFP (FFG) seeds imbibed at 21°C and 32°C for up to 72 h under constant light. WT in D) is shown as the negative control. FUS3-GFP (~MW 61 kDa) was detected with anti-GFP antibody. Comparable confocal settings were used in all images shown in B) and C). Duplicate experiments were conducted and one representative is shown.

Figure 3

Germination rates and seedling survival of FUS3 loss- and gain-of-function mutants during imbibition at supraoptimal temperature and recovery after heat stress. (A-G) Germination rates and% of seedlings survival of wild type (WT), FUS3 loss-of-function mutant (fus3-3) and ML1:FUS3-GFP (MFG) gain-of-function mutant during imbibition at control (21°C) and supraoptimal temperature (32°C). (A, B, E) Germination (radicle protrusion) rates of mature WT and MFG dry seeds imbibed on filter papers on MS plates in the absence or presence of 10 μM fluridone (+F) at 21°C (A) and 32°C (B). After six days of imbibition at 32°C, plates in B) were transferred to the control temperature (21°C) for six additional days and the% of seedlings survival (showing green cotyledon and green leaves) was calculated in (E). (C, D, F) Germination (radicle protrusion) rates of immature WT and fus3-3 green seeds imbibed on filter papers on MS media in the presence or absence of 10 μM fluridone (+F) at 21°C (C) and 32°C (D). After six days of imbibition at 32°C, plates in D) were transferred to the control temperature (21°C) for six additional days and the% of seedlings survival (showing green cotyledon and green leaves) was calculated in (F). (G) Appearance of representative WT and MFG seeds from the experiment shown in B). Averages from triplicates ± s.d. are shown (n ≥ 300 seeds). Experiments were repeated at least twice with similar results and one representative is shown. F, fluridone. Red bars, 32°C. Black bars, 21°C.

Figure 4

Cluster analysis of differentially expressed HSR genes. (A, B) Venn diagrams showing the number of transcripts with enhanced (A) or decreased (B) expression levels in seeds imbibed for 1, 12 and 24 h at 32°C compared to 21°C. Only genes showing a minimum fold change 1.8 (log₂ ≥ 0.85) and a p-value < 0.05 were selected. Results are presented as averages of two independent experiments. (C) Heat map of differentially expressed genes in seeds imbibed for 1, 12 and 24 h at 32°C compared to 21°C based on hierarchical clustering. Red color indicates upregulated genes, blue color indicates downregulated genes and yellow color indicates genes whose expression is unchanged.

Figure 5

Time-course expression analysis of HS-related and seed maturation genes. Expression patterns of select genes involved in heat-shock response (Heat Shock Protein, HSP; small HSP, sHSP; Heat Shock Factors, HSF) and late-embryogenesis abundant (LEA) and seed maturation (storage lipids and proteins) genes after 1, 12 and 24 h of imbibition at 32°C compared to 21°C. Y axis, fold-changes (log₂) in genes expression levels at 32°C compared to 21°C. × axis, hours (h) after imbibitions.

Figure 6

Time-course expression analysis of select HSR genes. Expression patterns of hormone (ABA and GA) metabolism and signaling genes after 1, 12 and 24 h of imbibition at 32°C compared to 21°C. Y axis, fold-changes (log₂) in genes expression levels at 32°C compared to 21°C. × axis, hours (h) after imbibitions.

Figure 7

Proposed model of thermoinhibition of seed germination. During seed germination at supraoptimal temperature, induction of ABA and repression of GA syntheses and signaling delays germination. FUS3 acts in a short developmental window to prevent seedling growth, by positively regulating ABA while negatively regulating GA biosyntheses. ABA increases FUS3 stability by positive feedback regulation.