Twilight length alters growth and flowering time in Arabidopsis via LHY/ CCA1

Abstract

Decades of research have uncovered how plants respond to two environmental variables that change across latitudes and over seasons: photoperiod and temperature. However, a third such variable, twilight length, has so far gone unstudied. Here, using controlled growth setups, we show that the duration of twilight affects growth and flowering time via the LHY/CCA1 clock genes in the model plant Arabidopsis. Using a series of progressively truncated no-twilight photoperiods, we also found that plants are more sensitive to twilight length compared to equivalent changes in solely photoperiods. Transcriptome and proteome analyses showed that twilight length affects reactive oxygen species metabolism, photosynthesis, and carbon metabolism. Genetic analyses suggested a twilight sensing pathway from the photoreceptors PHY E, PHY B, PHY D, and CRY2 through LHY/CCA1 to flowering modulation through the GI-FT pathway. Overall, our findings call for more nuanced models of day-length perception in plants and posit that twilight is an important determinant of plant growth and development.

Fig. 1.. The impact of twilight length on plant growth and development.

(A) Twilight length experimental schemes. (B) Leaf area of WT plants as measured using PlantCV from rosette images at 25 days after imbibition (n > 12). (C) Fresh weight of plants grown under different lengths of twilight (n > 38 plants) measured at 25 days after imbibition. (D and E) Flowering time measured by counting the number of days to bolting and the number of leaves at bolting respectively (n > 10 plants). (F) Representative images of plants at 36 days after imbibition. Letters above all graphs depict significantly different data points based on a one-way ANOVA and Tukey’s post hoc test with adjusted P < 0.05.

Fig. 2.. Dissecting the differences between plant responses to twilight and photoperiod.

(A) Depiction of the light treatments in this experiment from a 12-hour light:12-hour dark photoperiod (0sq) to 2- (2sq), 5- (5sq), 15- (15sq), and 30-min (30sq) reductions in photoperiod in the morning and evening (only morning shown). A 30-min twilight condition (30tw) is included as control. Dotted lines depict light intensity at different points along the twilight ramp. Flowering time measurements in terms of (B) days to bolting and (C) number of leaves at bolting of WT plants grown under the conditions depicted in (A). A minimum of 12 plants per treatment were measured. Letters depict significantly different data points based on a one-way ANOVA and Tukey’s post hoc test with adjusted P < 0.05.

Fig. 3.. lhy cca1 plants exhibit a muted phenotypic response to different twilight lengths.

(A) Leaf area of lhy cca1 plants at 25 days after imbibition measured using PlantCV (n > 12). (B) Fresh weight measurements of lhy cca1 plants at 25 days after imbibition (n > 39). (C and D) Flowering time measured in terms of (C) the number of leaves at bolting and (D) days to bolting (n > 22). Letters depict significantly different data points based on a one-way ANOVA and Tukey’s post hoc test with adjusted P < 0.05.

Fig. 4.. Proteome and transcriptome dynamics under different periods of twilight.

(A) Number of proteins that change significantly in abundance at each of the different twilight durations in lhy cca1 compared to WT plants. (B) Number of differentially expressed genes between lhy cca1 and WT plants at each twilight length. (C) Upset plot showing the overlaps in proteins significantly changing in abundance and (D) differentially expressed genes under different twilight durations (compared to no twilight) in both lhy cca1 and WT plants. (E) Shape-based clustering of protein abundance (z-score standardized) with clusters annotated with enriched Gene Ontology terms and selected member proteins and pathways. Blue lines show cluster centroids. Pie charts show the proportion of proteins in each cluster found in lhy cca1 (yellow) and WT (blue) samples. ROS, reactive oxygen species.

Fig. 5.. Proposed genetic pathway connecting twilight length to flowering time.

Genes depicted in color are genetically linked to twilight length sensing (via chronometric or morphometric flowering time changes, or both) according to this study, while grayed-out genes are not.