Alternative splicing regulation of the bHLH central exon in the XV subfamily of Arabidopsis thaliana bHLH transcription factors.

(A) Exon and intron gene distribution (left) and phylogeny (right; adapted from Leivar and Quail, 2011) of all members of the XV subfamily of Arabidopsis bHLH transcription factors. The bHLH subdomains are shown in blue. For all genes with the exception of HFR1, percentages indicate the proportion of the bHLH domain encoded in each exon. (B) PSI (Percent Spliced In) of the bHLH central exon using publicly available RNA-seq data covering several Arabidopsis thaliana tissues and environmental conditions (Supplemental Table 1). Blue dots indicate PSI<90, the generally considered cutoff for exon skipping. n.d., not detected.

Impact of heat treatment in etiolated seedlings.

(A-B) PSI (Percent Spliced In) quantification (left) from RT-PCR (right) of the PIF4 alternatively-spliced exon in seedlings grown in continuous dark for 3 days (d) and then transferred to 37 ºC (red) or white light (WL; yellow), or kept at 22 ºC for 3 (A) or 9 (B) additional hours (h) in darkness (D; gray). Data represent means ± SEM of biological triplicates. -RT, no reverse transcriptase. (C) Representative image of 3-day-old wild-type (WT) and pifq seedlings subjected or not to heat stress. Scale bar, 5 mm. (D) Boxplot representations of the cotyledon aperture (left) and hypocotyl length (right) of at least 35 WT seedlings grown in darkness for 3 days and then transferred to 37 ºC in darkness (red) or kept in the dark at 22 ºC (gray) for the indicated time in hours. Asterisks indicate statistically differences between medians (Mann Whitney test). Fluorescence of protochlorophyllide (Pchlide; 635 nm) (E; n=4) and percentage of photobleaching (F; n=3) in heat-stressed (red) or unstressed (gray) etiolated WT and pifq seedlings. Asterisks indicate statistically differences between averages (t-test). (G) RT-qPCR analysis of PIF4 transcript levels in WT seedlings grown as in (B). Values were normalized to PP2A and expression levels are expressed relative to the initial time point set at one. Data represent means ± SEM of biological triplicates, and different letters denote statistically significant differences under each condition (Tukey test; P<0.05). n.d., not detected. (A, D, E, F) *P<0.05, **P<0.01 and ***P<0.001.

Enhancing expression of the PIF4 long isoform at 37ºC reduces the impact of heat stress in etiolated seedlings.

(A) PSI (Percent Spliced In) quantification of the PIF4 alternatively-spliced exon in wild-type (WT), pif4 and PIF4-L seedlings grown in continuous dark for 3 days (d) and then transferred to 37 ºC for 3 additional hours (h) in darkness. Data represent means ± SEM of biological triplicates. (B) Protochlorophyllide (Pchlide; 635 nm) fluorescence (left; n=4) and percentage of photobleaching (right; n=3) in 3-day-old etiolated WT, pif4 and PIF4-L seedlings transferred to 37 ºC (red) or kept at 22 ºC (gray) for 24 additional hours (h) in the dark. For photobleaching quantification, seedlings were subsequently exposed to white light (WL) for 3 days. Data represent means ± SEM of biological replicates (t-test). a.u., arbitrary units. (C) Boxplot representations of the cotyledon aperture (left) and hypocotyl length (right) of at least 35 WT, pif4 and PIF4-L seedlings grown as in (B). Mann Whitney test was used to define statistically differences. (D) Venn diagram showing overlap among heat-regulated genes in WT seedlings defined in this study and PIF-regulated and PIF-bound genes defined previously (Pfeiffer et al., 2014) (two-sided Fisher’s exact test). (E) Heat responsiveness (fold change; FC) in WT, pif4 and PIF4-L for heat-regulated genes in pif4 seedlings (n=3). Different letters denote statistically significant differences between genotypes by Dunn’s test (P<0.05). (A-C) Asterisks indicate statistically significant differences from pif4 (*P<0.05, **P<0.01 and ***P<0.001; n.s., not significant), and n the number of biological replicates.

Enhancing expression of the PIF4 short isoform promotes cotyledon opening in the dark.

(A) RT-qPCR analysis of PIF4 transcript levels in wild-type (WT), pif4 and PIF4-S seedlings grown for 4 days in darkness. Values were normalized to PP2A and expression levels are expressed relative to the WT. Data represent means ± SEM of technical triplicates. (B) RT-PCR of the alternatively-spliced exon of PIF4 in seedlings grown as in (A). -RT, no reverse transcriptase. (C) Boxplot representations of the cotyledon aperture (top) and hypocotyl length (bottom) of at least 28 WT, pif4 and PIF4-S seedlings grown for 3 or 4 days (d) in the dark (D). Asterisks indicate statistically differences from WT at each day (Mann Whitney test; *P<0.05, **P<0.01 and ***P<0.001; n.s., not significant).