Light and Alternating Temperatures Release Seed Dormancy in the Invasive Dipsacus fullonum L. Through ROS Homeostasis and ABA Regulation

Abstract

Seeds have developed mechanisms to perceive environmental signals, such as light and temperature, which govern germination and enhance the chance of seedling establishment. This study examined the foundations of light and temperature sensitivity in the seed dormancy release of a common weed, Dipsacus fullonum. By screening six accessions from two different regions, we identified two unique germination behaviors: one sensitive to environmental stimuli and one neutral to them. In the sensitive accession, ABA is crucial for regulating dormancy release, as it accumulates in seeds subjected to darkness, while it decreases under other conditions. We observed a rise of reactive oxygen species (ROS) under conditions that stimulate germination and highlighted that their presence enhanced germination even in the absence of light. This study employed a long-read RNA-seq technology to examine the regulation of key genes associated with germination. We identified the essential nodes in this process: DfPIF1, which maintains the dormancy state in darkness at constant temperatures mainly by promoting ABA biosynthesis and signaling, and antioxidant enzymatic machinery, DfMSD1, DfCSD2, DfAPX, and DfPRX1, whose activity regulates ROS homeostasis, promoting or inhibiting germination. This study provides novel mechanisms that regulate seed germination in weeds, specifically involving ABA regulation and ROS in response to environmental stimuli.

Keywords: PIF1; ROS scavengers; common teasel; environmental cues; histone deacetylase; long‐reads RNA‐seq; seed germination; weeds.

FIGURE 1

Germination behavior of different D. fullonum accessions: SIM, LAP, ETN, BAH, PIE, and LOM. Mean final germination (%) of seeds imbibed at constant temperatures (A) or at alternating temperatures (B) in L/D (yellow bars) (12 h of photoperiod) or in continuous dark (violet bars). Error bars represent confidence intervals calculated over 150 seeds.

FIGURE 2

Seed germination of LOM seeds with red or far‐red pulse irradiation. Left panel, irradiation treatments used for the experiment. Seeds were imbibed at the indicated temperatures in the L/D 12 h photoperiod (light grey) or in the continuous dark (dark grey) or with a preliminary exposure to far‐red (purple rectangles) or red light (red rectangles). Small rectangles indicate 5 min of exposition, while large ones indicate 24 h of exposition. Right panel, mean final germination percentage of seeds imbibed at the corresponding conditions described in the left panel. Error bars represent confidence intervals calculated over 150 seeds for each treatment.

FIGURE 3

Comparison of the effect of the addition of a ROS donor or inhibitors on germination of LOM seeds. Mean final germination of LOM seeds imbibed in the dark at constant temperature in the presence of MV (uppermost panel) or imbibed in the light at constant temperature with NAC (central panel) or DPI (lowermost panel) at different concentrations (or no treatment: CTRL). Error bars represent confidence intervals calculated over 150 seeds for each treatment.

FIGURE 4

In situ localization of superoxide anions (NBT) and H₂O₂ (DAB) in excised embryos of LOM accession. (A) NBT staining in seeds imbibed in the dark, or in the light (B), or at alternating temperatures (C), respectively. (D) DAB staining in seeds imbibed in the dark, or in the light (E), or at alternating temperatures (F), respectively.

FIGURE 5

Seed germination response in the presence of gibberellins, fluridone, or ABA and quantification of ABA. Time course germination of LOM seeds imbibed at 15°C in the dark with water (CTRL) or with 50, 100, or 200 μM of GA₄₊₇ (A) or with 1, 10, or 50 μM of fluridone (FLU) (B). Final germination percentage means of LOM seeds in the presence of 1, 5, 10, 25, 50, or 100 μM of ABA incubated in L/D at 15°C or 10°C/20°C (C). Error bars represent confidence intervals calculated over 150 seeds for each treatment. (D) ABA concentrations in ng g⁻¹ dry weight (DW) in LOM dry seeds (green), seeds imbibed at constant temperature in light (light blue), in darkness (black), in darkness after MV treatment (grey) and in darkness at alternating temperatures (purple). Error bars represent standard deviation.

FIGURE 6

Time course germination of LOM seeds imbibed with 10 mM of valproic acid (VA) or water (CTRL) in the presence/absence of light at constant (upper panel) or alternating temperatures (lower panel). Error bars represent confidence intervals calculated over 150 seeds for each condition.

FIGURE 7

Analysis of transcript assemblies. The left panel displays the BUSCO completeness assessment for conserved orthologous genes in the combined, de novo, and genome reference‐guided transcriptomes. Light blue: Complete single‐copy; pink: Complete duplicated; light green: Fragmented; light orange: Missing genes. On the right, MA plots showing the DEGs identified in the three comparisons (DCT vs. DAT, DCT vs. LCT and DCT vs. LAT). Fold change > 2, FDR < 0.01.

FIGURE 8

Analysis of identified DEGs. On the left panel, the upset plots of the overlapping DEGs across multiple comparisons. On the right, the bubble plot of top GO enrichment analysis showing the over‐represented terms in the three comparisons (DCT vs. DAT, DCT vs. LCT and DCT vs. LAT); the full list is provided as Set S2.

FIGURE 9

RT‐qPCR analyses of selected genes. LOM seeds imbibed at constant temperature in light (light blue), in the dark (dashed bar), or at alternating temperatures in the dark (purple). The expression levels are presented as fold change relative to the average expression level in the dark at constant temperature, which is represented by a dashed bar. Significance level: p < 0.05; p < 0.01 **; p < 0.001 ***.

FIGURE 10

Proposed model for the regulation of seed dormancy release in D. fullonum . Left panel (dark, constant temperature): DfPIF1 and genes related to ROS‐scavenging (DfPRX, DfCSD, DfMSD1, DfGSTU) are upregulated. PIF1 promotes ABA biosynthesis and signaling, while antioxidant activity maintains low ROS levels. Together, these mechanisms reinforce the dormant state. Central panel (light, constant temperature): DfPIF1 is downregulated, whereas antioxidant enzymes and HDAs are upregulated. Reduced ABA biosynthesis/signaling and controlled ROS accumulation favor GA biosynthesis (DfGA3OX) and cell wall loosening (DfXTH9), leading to dormancy release. Right panel (dark, alternating temperatures): Both DfPIF1 and antioxidant enzymes are downregulated, resulting in higher ROS accumulation compared to constant darkness. Increased ROS levels promote ABA catabolism, while HDA inhibition of ABA‐responsive genes further contributes to dormancy release. Arrows indicate positive regulation, bars indicate negative regulation; solid lines represent direct interactions, dashed lines represent indirect ones. Upregulated factors are shown with larger icons and labels, while downregulated factors are shown with smaller icons and labels.