ZEITLUPE positively regulates hypocotyl elongation at warm temperature under light in Arabidopsis thaliana

Abstract

Hypocotyl cell elongation has been studied as a model to understand how cellular expansion contributes to plant organ growth. Hypocotyl elongation is affected by multiple environmental factors, including light quantity and light quality. Red light inhibits hypocotyl growth via the phytochrome signaling pathways. Proteins of the FLAVIN-BINDING KELCH REPEAT F-BOX 1 / LOV KELCH PROTEIN 2 / ZEITLUPE family are positive regulators of hypocotyl elongation under red light in Arabidopsis. These proteins were suggested to reduce phytochrome-mediated inhibition of hypocotyl elongation. Here, we show that ZEITLUPE also functions as a positive regulator in warmth-induced hypocotyl elongation under light in Arabidopsis.

Abbreviations

FKF1

FLAVIN-BINDING KELCH REPEAT F-BOX 1

LKP2

LOV KELCH PROTEIN 2

PIF

PHYTOCHROME-INTERACTING FACTOR

phyB

phytochrome B

ZTL

ZEITLUPE

Plants use light not only as an energy source, but also as an environmental signal for growth and development. Among numerous genes and proteins involved in the perception and transduction of environmental signals, those for photoreceptors are well investigated.^1,2

The FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) / LOV KELCH PROTEIN 2 (LKP2) / ZEITLUPE (ZTL) family is a group of blue-light photoreceptors in Arabidopsis.^3–6 These proteins determine the period of circadian oscillation, regulate photoperiodic flowering, and are involved in light-controlled hypocotyl elongation.^3–6 fkf1 mutants have short hypocotyls under continuous blue or red light.⁴ LKP2-overproducing plants have elongated hypocotyls under continuous blue, red, or white light.⁵ ztl mutants are indistinguishable from wild type under blue light but have short hypocotyls under continuous red light;⁶ ZTL-overproducing plants have elongated hypocotyls under blue, red, or white light.⁴ These results suggest that, even though they are blue-light photoreceptors, proteins of this family promote hypocotyl growth under red or white light by inhibiting the phytochrome B (phyB)–mediated signal transduction pathway, as phyB is the main receptor mediating red light–induced inhibition of hypocotyl elongation.^1,2

Hypocotyl growth is affected by both light and temperature. The hypocotyls of Arabidopsis plants grown at 28°C are longer than those grown at 22°C.⁷ The basic helix-loop-helix transcriptional regulator PHYTOCHROME-INTERACTING FACTOR (PIF) 4 functions as a positive regulator in this temperature-mediated hypocotyl elongation, as indicated by the fact that the pif4 mutant does not show enhanced hypocotyl elongation at 28°C under continuous light.⁷ PIF4 is destabilized by photoactivated phyB, indicating that the stability of PIF4 is modulated by light.⁸ These facts suggest that signals from light and temperature are integrated at PIF4 for hypocotyl growth regulation. Therefore, factors that inhibit phyB signaling, such as FKF1/LKP2/ZTL family proteins, may promote temperature-mediated hypocotyl elongation.

To assess this possibility, we grew ztl-3, ztl-105, fkf1-t lkp2-1 ztl-105,⁹ and Columbia seedlings under continuous white light (80 μmol·m^–2·s^–1) or in the dark on 1/2-basal-salt Murashige and Skoog agar (0.8% w/v) at 22 or 28°C, and measured the hypocotyl length as described previously (Fig. 1A–D).¹⁰ At 22°C under continuous white light, hypocotyls of the mutants were slightly but significantly shorter than those of the wild type (by 25% for ztl-3, 19% for ztl-105, and 29% for fkf1-t lkp2-1 ztl-105; Fig. 1A). At 28°C under the same light conditions (Fig. 1C), hypocotyls of the wild type and mutants were longer than at 22°C (Fig. 1A): ×3.1 for wild type but only ×2.2 for both ztl3 and ztl-105 and ×2.0 for fkf1-t lkp2-1 ztl-105. There were no statistically significant differences between the hypocotyl lengths of dark-grown wild-type and mutant plants either at 22°C (Fig. 1B) or at 28°C (Fig. 1D), indicating that the differences in hypocotyl length between wild type and mutants were light dependent.

Figure 1.

Hypocotyl length of Arabidopsis thaliana plants grown under different temperature and light conditions. Seedlings of control wild-type Columbia (Col) plants and 3 mutants (ztl-3, ztl-105, and fkf1-t lkp2-1 ztl-105) were grown on 1/2-basal-salt Murashige and Skoog agar (0.8% w/v) medium. (A, B) Hypocotyl length of seedlings grown for 8 days at 22°C under continuous white light (80 μmol·m^–2·s^–1) (A) or in the dark (B). (C, D) Hypocotyl length of seedlings grown for 3 days at 22°C and then for 5 days at 28°C under continuous white light (80 μmol·m^–2·s^–1) (C) or in the dark (D). (E, F) A photograph and hypocotyl length of seedlings grown for 3 days at 22°C and then for 5 days at 28°C under continuous red light (10 μmol·m^–2·s^–1). Error bars represent standard error of the mean (n = 7–27); *P<0.05 and **P <0.001 (Student's t-test) in comparison with Col. Scale bar = 5 mm.

Next, we grew ztl-3, ztl-105, fkf1-t lkp2-1 ztl-105, and Columbia seedlings on the same medium under continuous red light (10 μmol·m^–2·s^–1) at 28°C (Fig. 1E, F). Hypocotyls of the mutants were significantly shorter than those of the wild-type seedlings (by 57% for ztl-3 and ztl-105 and by 59% for fkf1-t lkp2-1 ztl-105; Fig. 1F). The differences in hypocotyl length between ztl and fkf1 lkp2 ztl mutants were small at 28°C under either continuous white or red light. These data suggest that, among FKF1/LKP2/ZTL family members, ZTL is the major contributor to hypocotyl elongation at 28°C under white or red light in wild-type plants.

How does ZTL reduce light-induced hypocotyl growth inhibition and promote warm temperature–mediated hypocotyl elongation? Upon absorbing red light, light-activated phyB enters the nucleus, where it interacts with several regulatory proteins, including PIFs, and modulates their activity or stability, thereby inducing light responses by altering the expression of various genes.¹¹ Dark-grown pif mutants show constitutive photomorphogenic phenotypes, including short hypocotyls, as though they grew in red light; PIFs therefore function negatively in this phytochrome-mediated red light signaling.^12,13 Under red light, PIF4 accumulates in seedlings in response to warm temperatures.¹⁴ In response to warm temperature under white light, PIF4 binds to promoter regions of 2 genes for auxin biosynthesis enzymes, TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 and CYP79B2, promoting gene expression, which leads to elevated indole-3-acetic acid levels in seedlings and thus to hypocotyl elongation.¹⁵ Light-activated phyB mediates PIF4 degradation by the proteasome.⁸ ZTL and PHYB apoprotein can interact.¹⁶ Interaction between ZTL and phyB might inhibit that between phyB and PIF4 and thus increase the level of free PIF4. Inhibition of the phyB–PIF4 interaction by ZTL could explain the role of ZTL in the promotion of warm temperature–induced hypocotyl elongation under red light if ZTL level, activity, or both are increased in response to elevated temperatures. In this respect, it may be relevant that heat shock protein HSP90 has been reported to be essential for ZTL accumulation and function.¹⁷

Further investigation is necessary to assess this possibility and understand how ZTL regulates hypocotyl growth.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This research was partially supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Asuka Sugitani and Hiroyuki Kitaki for their technical assistance.