ZTL regulates thermomorphogenesis through TOC1 and PRR5.

Plants adapt to high temperature stresses through thermomorphogenesis, a process that includes stem elongation and hyponastic leaf growth. Thermomorphogenesis is gated by the circadian clock through two evening-expressed clock components, TIMING OF CAB EXPRESSION1 (TOC1) and PSEUDO-RESPONSE REGULATORS5 (PRR5). These proteins directly interact with and inhibit PHYTOCHROME INTERACTING FACTOR4 (PIF4), a basic helix-loop-helix transcription factor that promotes thermoresponsive growth. PIF4-mediated thermoresponsive growth is positively regulated by ZEITLUPE (ZTL), a central clock component, but the molecular mechanisms underlying this are poorly understood. Here, we show that ZTL regulates thermoresponsive growth through TOC1 and PRR5. Genetic analyses reveal that ZTL regulates PIF4 activity as well as PIF4 expression. In Arabidopsis thaliana, ztl mutants exhibit highly accumulated TOC1 and PRR5 and unresponsive expression of PIF4 target genes under exposure to high temperatures. Mutations in TOC1 and PRR5 restore thermoactivation of PIF4 target genes and thermoresponsive growth in ztl mutants. We also show that the molecular chaperone heat-shock protein 90 promotes thermoresponsive growth through the ZTL-TOC1/PRR5 signaling module. Further, we show that ZTL protein stability is increased at high temperatures. Taken together, our results demonstrate that ZTL-mediated degradation of TOC1 and PRR5 enhances the sensitivity of hypocotyl growth to high temperatures.

Overexpression of BBX18 Promotes Thermomorphogenesis Through the PRR5-PIF4 Pathway.

Thermomorphogenesis is the morphological response of plants to an elevation in the ambient temperature, which is mediated by the bHLH transcription factor PIF4. The evening-expressed clock component, PRR5, directly represses the expression of PIF4 mRNA. Additionally, PRR5 interacts with PIF4 protein and represses its transactivation activity, which in turn suppresses the thermoresponsive growth in the evening. Here, we found that the B-box zinc finger protein, BBX18, interacts with PRR5 through the B-Box2 domain. Deletion of the B-Box2 domain abolished the functions of BBX18, including the stimulation of PIF4 mRNA expression and hypocotyl growth. Overexpression of BBX18, and not of B-Box2-deleted BBX18, restored the expression of thermoresponsive genes in the evening. We further show that BBX18 prevents PRR5 from inhibiting PIF4-mediated high temperature responses. Taken together, our results suggest that BBX18 regulates thermoresponsive growth through the PRR5-PIF4 pathway.

Chemical control of receptor kinase signaling by rapamycin-induced dimerization.

Membrane-localized leucine-rich repeat receptor kinases (LRR-RKs) sense diverse extracellular signals, and coordinate and specify cellular functions in plants. However, functional understanding and identification of the cellular signaling of most LRR-RKs remain a major challenge owing to their genetic redundancy, the lack of ligand information, and subtle phenotypes of LRR-RK overexpression. Here, we report an engineered rapamycin-inducible dimerization (RiD) receptor system that triggers a receptor-specific LRR-RK signaling independent of their cognate ligands or endogenous receptors. Using the RiD-receptors, we demonstrated that the rapamycin-mediated association of chimeric cytosolic kinase domains from the BRI1/BAK1 receptor/co-receptor, but not the BRI1/BRI1 or BAK1/BAK1 homodimer, is sufficient to activate downstream brassinosteroid signaling and physiological responses. Furthermore, we showed that the engineered RiD-FLS2/BAK1 could activate flagellin-22-mediated immune signaling and responses. Using the RiD system, we also identified the potential function of an unknown orphan receptor in immune signaling and revealed the differential activities of SERK co-receptors of LRR-RKs. Our results indicate that the RiD method can serve as a synthetic biology tool for precise temporal manipulation of LRR-RK signaling and for understanding LRR-RK biology.

The epidermis coordinates thermoresponsive growth through the phyB-PIF4-auxin pathway.

In plants, an elevation in ambient temperature induces adaptive morphological changes including elongated hypocotyls, which is predominantly regulated by a bHLH transcription factor, PIF4. Although PIF4 is expressed in all aerial tissues including the epidermis, mesophyll, and vascular bundle, its tissue-specific functions in thermomorphogenesis are not known. Here, we show that epidermis-specific expression of PIF4 induces constitutive long hypocotyls, while vasculature-specific expression of PIF4 has no effect on hypocotyl growth. RNA-Seq and qRT-PCR analyses reveal that auxin-responsive genes and growth-related genes are highly activated by epidermal, but not by vascular, PIF4. Additionally, inactivation of epidermal PIF4 or auxin signaling, and overexpression of epidermal phyB suppresses thermoresponsive growth, indicating that epidermal PIF4-auxin pathways are essential for the temperature responses. Further, we show that high temperatures increase both epidermal PIF4 transcription and the epidermal PIF4 DNA-binding ability. Taken together, our study demonstrates that the epidermis regulates thermoresponsive growth through the phyB-PIF4-auxin pathway.

Trehalose-6-phosphate signaling regulates thermoresponsive hypocotyl growth in Arabidopsis thaliana.

Growth plasticity is a key mechanism by which plants adapt to the ever-changing environmental conditions. Since growth is a high-energy-demanding and irreversible process, it is expected to be regulated by the integration of endogenous energy status as well as environmental conditions. Here, we show that trehalose-6-phosphate (T6P) functions as a sugar signaling molecule that coordinates thermoresponsive hypocotyl growth with endogenous sugar availability. We found that the loss of T6P SYNTHASE 1 (TPS1) in Arabidopsis thaliana impaired high-temperature-mediated hypocotyl growth. Consistently, the activity of PIF4, a transcription factor that positively regulates hypocotyl growth, was compromised in the tps1 mutant. We further show that, in the tps1 mutant, a sugar signaling kinase KIN10 directly phosphorylates and destabilizes PIF4. T6P inhibits KIN10 activity in a GRIK-dependent manner, allowing PIF4 to promote hypocotyl growth at high temperatures. Together, our results demonstrate that T6P determines thermoresponsive growth through the KIN10-PIF4 signaling module. Such regulation of PIF4 by T6P integrates the temperature-signaling pathway with the endogenous sugar status, thus optimizing plant growth response to environmental stresses.

High Ambient Temperature Represses Anthocyanin Biosynthesis through Degradation of HY5.

Anthocyanins are flavonoid compounds that protect plant tissues from many environmental stresses including high light irradiance, freezing temperatures, and pathogen infection. Regulation of anthocyanin biosynthesis is intimately associated with environmental changes to enhance plant survival under stressful environmental conditions. Various factors, such as UV, visible light, cold, osmotic stress, and pathogen infection, can induce anthocyanin biosynthesis. In contrast, high temperatures are known to reduce anthocyanin accumulation in many plant species, even drastically in the skin of fruits such as grape berries and apples. However, the mechanisms by which high temperatures regulate anthocyanin biosynthesis in Arabidopsis thaliana remain largely unknown. Here, we show that high ambient temperatures repress anthocyanin biosynthesis through the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and the positive regulator of anthocyanin biosynthesis ELONGATED HYPOCOTYL5 (HY5). We show that an increase in ambient temperature decreases expression of genes required in both the early and late steps of the anthocyanin biosynthesis pathway in Arabidopsis seedlings. As a result, seedlings grown at a high temperature (28°C) accumulate less anthocyanin pigment than those grown at a low temperature (17°C). We further show that high temperature induces the degradation of the HY5 protein in a COP1 activity-dependent manner. In agreement with this finding, anthocyanin biosynthesis and accumulation do not respond to ambient temperature changes in cop1 and hy5 mutant plants. The degradation of HY5 derepresses the expression of MYBL2, which partially mediates the high temperature repression of anthocyanin biosynthesis. Overall, our study demonstrates that high ambient temperatures repress anthocyanin biosynthesis through a COP1-HY5 signaling module.

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

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.