PIF4 Promotes Expression of LNG1 and LNG2 to Induce Thermomorphogenic Growth in Arabidopsis

Abstract

Arabidopsis plants adapt to high ambient temperature by a suite of morphological changes including elongation of hypocotyls and petioles and leaf hyponastic growth. These morphological changes are collectively called thermomorphogenesis and are believed to increase leaf cooling capacity by enhancing transpiration efficiency, thereby increasing tolerance to heat stress. The bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) has been identified as a major regulator of thermomorphogenic growth. Here, we show that PIF4 promotes the expression of two homologous genes LONGIFOLIA1 (LNG1) and LONGIFOLIA2 (LNG2) that have been reported to regulate leaf morphology. ChIP-Seq analyses and ChIP assays showed that PIF4 directly binds to the promoters of both LNG1 and LNG2. The expression of LNG1 and LNG2 is induced by high temperature in wild type plants. However, the high temperature activation of LNG1 and LNG2 is compromised in the pif4 mutant, indicating that PIF4 directly regulates LNG1 and LNG2 expression in response to high ambient temperatures. We further show that the activities of LNGs support thermomorphogenic growth. The expression of auxin biosynthetic and responsive genes is decreased in the lng quadruple mutant, implying that LNGs promote thermomorphogenic growth by activating the auxin pathway. Together, our results demonstrate that LNG1 and LNG2 are directly regulated by PIF4 and are new components for the regulation of thermomorphogenesis.

Keywords: Arabidopsis; ChIP; LNG; PIF4; gene expression; high temperature stress; thermomorphogenesis.

FIGURE 1

PHYTOCHROME INTERACTING FACTOR4 directly binds to the promoters of LNG1 and LNG2. (A) Previous ChIP-Seq analyses (Oh et al., 2012) showing the PIF4 binding peaks around the promoters of LNG1 and LNG2. (B) A diagram of the LNG1 and LNG2 promoters showing the positions of PBE (PIF4 Binding E-box: CATGTG) motifs. a, b, and, c indicate the ChIP amplicons in the ChIP assays in (C). (C) ChIP-quantitative PCR assays of PIF4 binding to LNG1 and LNG2 promoters. Five-day-old WT and PIF4p::PIF4-MYC transgenic seedlings were grown at 20°C, then switched to 28°C for 4 h or kept at 20°C; the seedlings were then harvested for ChIP assays using an anti-MYC antibody. Enrichment of DNA was calculated as the ratio between PIF4p::PIF4-MYC and WT control, normalized to that of the PP2A coding region as an internal reference. Error bars indicate standard deviation (SD, n = 3).

FIGURE 2

High temperature activates the expression of LNG1 and LNG2 in a PIF4 dependent manner. (A) Diagram showing plant growth conditions used for the qRT-PCR analyses in (B) and (F). Seedlings were maintained under 12 h light/12 h dark conditions at 20°C for 4 days, and then transferred to 24 h light conditions. The seedlings were then exposed for 1 or 4 h to high temperature (28°C) from ZT20 and harvested for RNA extraction. (B–D) The qRT-PCR analysis of the expression levels of PIF4, LNG1, and LNG2 in WT seedlings under the indicated growth conditions. Gene expression levels were normalized to PP2A and presented as values relative to those of the WT seedlings at 20°C at ZT20. Error bars indicate standard deviation (SD, n = 3). (E,F) The qRT-PCR analysis of the expression levels of LNG1 and LNG2. WT and pif4 mutant seedlings were exposed for 4 h to high temperature (28°C) from ZT20 or kept at 20°C. Error bars indicate SD (n = 3). Different letters above each bar indicate statistically significant differences (ANOVA and Tukey’s HSD; P < 0.05).

FIGURE 3

LONGIFOLIA activities are required for thermomorphogenic growth. (A) Diagram showing plant growth conditions for hypocotyl length measurements. WT and lngq seedlings were grown at 20°C for 7 days or grown at 20°C for 4 days followed by incubation at 28°C for 3 days before harvesting for hypocotyl length measurements. (B) Representative seedlings grown under the conditions described in (A). (C) Average hypocotyl lengths of WT and lngq seedlings grown under the conditions described in (A). Error bars indicate standard deviation (SD, n = 10 plants). Numbers indicate ratios of hypocotyl lengths (28°C/20°C). Different letters above each bar indicate statistically significant differences (ANOVA and Tukey’s HSD; P < 0.05). (D) Representative leaf blades and petioles of wild type and lngq plants grown at 20°C or 28°C for 2 weeks. Bar indicates 5 mm. (E) Average petiole lengths of WT and lngq plants grown at two different temperatures. Error bars indicate SD (n = 20 petioles). Numbers indicate ratios of petiole lengths (28°C/20°C). Different letters above each bar indicate statistically significant differences (ANOVA and Tukey’s HSD; P < 0.05). (F) Average ratio of the leaf length to width of plants grown at 20°C or 28°C. The third and fourth leaves were analyzed. Error bars indicate SD (n = 10 leaves). ^∗P < 0.05 (Student’s t-test) and ns indicates not significant.

FIGURE 4

The expression of YUC8 and IAA29 is hyposensitive to high temperature in lngq seedlings. (A) Diagram showing plant growth conditions used for the qRT-PCR analyses in (B–D). Seedlings were maintained under 12 h light/12 h dark at 20°C for 4 days, and then transferred to 24 h light conditions. The seedlings were then exposed for 4 h to high temperature (28°C) at ZT20 and harvested for RNA extraction. (B–D) The qRT-PCR analysis of the expression level of YUC8 and IAA29. Gene expression levels were normalized to PP2A and presented as values relative to those of the WT seedlings at 20°C. Error bars indicate standard deviation (SD, n = 3). Numbers indicate ratios of the expression levels (28°C/20°C). Different letters above each bar indicate statistically significant differences (ANOVA and Tukey’s HSD; P < 0.05). (E) Western blotting with anti-PIF4 antibody showed that PIF4 protein levels were increased by high temperatures in both wild type and lngq seedlings. Total protein was extracted from the seedlings grown in the conditions described in (A). Equal loading of samples is shown by Ponceau S staining. (F) A hypothetical model depicting PIF4-LNGs-mediated thermomorphogenic growth. At elevated temperatures, PIF4 transcription factor binds to the promoters of LNG1 and LNG2 as well as auxin-related genes (YUC8 and IAA29), and activate their expression. The increased LNG proteins further activate the expression of YUC8 and IAA29 through unknown mechanisms. The resulting increased auxin biosynthesis and signaling induce thermomorphogenic growth.