Phytochrome-interacting factor 4 (PIF4) regulates auxin biosynthesis at high temperature

Abstract

At high ambient temperature, plants display dramatic stem elongation in an adaptive response to heat. This response is mediated by elevated levels of the phytohormone auxin and requires auxin biosynthesis, signaling, and transport pathways. The mechanisms by which higher temperature results in greater auxin accumulation are unknown, however. A basic helix-loop-helix transcription factor, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), is also required for hypocotyl elongation in response to high temperature. PIF4 also acts redundantly with its homolog, PIF5, to regulate diurnal growth rhythms and elongation responses to the threat of vegetative shade. PIF4 activity is reportedly limited in part by binding to both the basic helix-loop-helix protein LONG HYPOCOTYL IN FAR RED 1 and the DELLA family of growth-repressing proteins. Despite the importance of PIF4 in integrating multiple environmental signals, the mechanisms by which PIF4 controls growth are unknown. Here we demonstrate that PIF4 regulates levels of auxin and the expression of key auxin biosynthesis genes at high temperature. We also identify a family of SMALL AUXIN UP RNA (SAUR) genes that are expressed at high temperature in a PIF4-dependent manner and promote elongation growth. Taken together, our results demonstrate direct molecular links among PIF4, auxin, and elongation growth at high temperature.

Fig. 1.

PIF4 regulates IAA abundance and activity at high temperature. (A) IAA concentration in 6-d-old seedlings grown in continuous light at 20 °C (20), 20 °C and 24 h at 28 °C treatment (20 + 28), or 28 °C (28). Bars represent SE. P < 0.01 (B) GUS transcript abundance in 6-d-old Col[DR5-GUS] and pif4/DR5-GUS seedlings grown in continuous light at 20 °C (20) or for 5 d at 20 °C and and 24 h at 28 °C (28). Data are from three biological repeats. Bars represent SE (P < 0.05). (C) Hypocotyl lengths of 9-d-old WT and pif4-101 seedlings grown at 28 °C with and without the synthetic auxin picloram. Seedlings were grown in continuous light at 20 °C for 2 d before transfer to plates containing 0.1 μM picloram at 28 °C. Bars represent SE. P < 0.05.

Fig. 2.

PIF4 regulates the auxin biosynthesis genes TAA1 and CYP79B2 and binds to their promoters in a temperature-dependent manner. (A and B) TAA1 (A) and CYP79B2 (B) transcript abundance in 6-d-old WT and pif4-101 seedlings grown in continuous light at 20 °C (20) or for 5 d at 20 °C and 24 h at 28 °C (28). Values represent the means of three biological repeats. Bars represent SE. P < 0.05. (C) Position of G-boxes and E-boxes in TAA1 and CYP79B2. TSS, transcriptional start site. (D and E) ChIP analysis of 35S::PIF4:HA plants showing enhanced binding of PIF4 to TAA1 (D) and CYP79B2 (E) at high temperature. Plants were grown at 12 °C for 7 d (12) or at 12 °C for 5 d then at 27 °C for 48 h (27). Multiple regions of each gene promoter and upstream coding sequence were analyzed, as shown in C. Mock reactions were performed without antibody. Data are from three biological repeats. Bars represent SE. a, significant PIF4 enrichment compared with the respective mock samples (P < 0.05); b, Significant PIF4 enrichment compared with the respective mock samples (P < 0.005). *Increased PIF4 binding at 27 °C compared with 12 °C (P < 0.05). ^#Increased PIF4 binding at 27 °C compared with 12 °C (P < 0.005).

Fig. 3.

PIF4 regulates expression of the SAUR19–24 subfamily at high temperature. (A) Relative transcript abundance of SAUR genes 19–24 in 6-d-old Col-0 and pif4-101 seedlings grown continuously at 20 °C (20) or grown at 20 °C and shifted to 28 °C for 6 h (28). Data represent the means of three biological repeats. SE values are shown. P < 0.05. (B) PIF4-regulated SAUR genes are strongly expressed in seedling hypocotyls at high temperature. GUS staining of 6-d-old seedlings expressing pSAUR19-GUS, pSAUR21-GUS, pSAUR23-GUS, and pSAUR 24-GUS grown in continuous light at 20 °C and 28 °C, and pif4[pSAUR19-GUS] and pif4[pSAUR24-GUS] seedlings grown at 20 °C and 28 °C, is shown. (Scale bars: 1 mm.) All seedlings were stained for the same duration.

Fig. 4.

Overexpression of SAUR19 rescues the pif4 phenotype at high temperature. Hypocotyl lengths of WT, pif4-101, and GFP-SAUR19 overexpression lines in WT and pif4-101 backgrounds. Seedlings were grown for 6 d in continuous light at 20 °C and 28 °C. Bars represent SE. P < 0.05.