Type B Response Regulators Act As Central Integrators in Transcriptional Control of the Auxin Biosynthesis Enzyme TAA1

Abstract

During embryogenesis and organ formation, establishing proper gradient is critical for auxin function, which is achieved through coordinated regulation of both auxin metabolism and transport. Expression of auxin biosynthetic genes is often tissue specific and is regulated by environmental signals. However, the underlying regulatory mechanisms remain elusive. Here, we investigated the transcriptional regulation of a key auxin biosynthetic gene, l-Tryptophan aminotransferase of Arabidopsis1 (TAA1). A canonical and a novel Arabidopsis (Arabidopsis thaliana) response regulator (ARR) binding site were identified in the promoter and the second intron of TAA1, which were required for its tissue-specific expression. C-termini of a subset of the type B ARRs selectively bind to one or both cis elements and activate the expression of TAA1 We further demonstrated that the ARRs not only mediate the transcriptional regulation of TAA1 by cytokinins, but also mediate its regulation by ethylene, light, and developmental signals. Through direct protein-protein interactions, the transcriptional activity of ARR1 is enhanced by ARR12, DELLAs, and ethylene-insenstive3 (EIN3). Our study thus revealed the ARR proteins act as key node that mediate the regulation of auxin biosynthesis by various hormonal, environmental, and developmental signals through transcriptional regulation of the key auxin biosynthesis gene TAA1.

Figure 1.

Identification of the regulatory elements that control tissue-specific expression of TAA1. A, GUS expression patterns of 5-d-old TAA1 _pro:TAA1g-GUS and TAA1_pro:GUS seedlings. B, Schematic diagram showing DNA fragments used for cis-element analysis. C, GUS expression patterns of 5-d-old P1-CORE_pro:GUS and P5-CORE_pro:GUS seedlings.

Figure 2.

Interactions between ARRs and P1/P5 in vitro. A, Interactions among ARR1C, ARR10C, ARR12C, and P1/P5 in yeast. pABAi, empty vector control. ABA: Aureobasidin A; AD:pGADT7. B, Partial sequences of P1b2, P5b1, P5b3, and their corresponding mutant forms. Predicted ARR binding sites and their corresponding mutant forms were italicized and underlined. C, Y1H results showing interactions between ARR1C and P1/P5 were abolished by mutations in the putative ARR binding sites. D, EMSA assay showing ARR1C binds specifically to P1b2, P5b1, and P5b3, His-ARR1C:His-tagged ARR1C. Probe and the mutant competitor (Mu) sequences are shown at the bottom. The putative ARR binding sites and their corresponding mutant forms were italicized and underlined. Arrowheads mark the shifted bands.

Figure 3.

ARR1C binds to both P1 and P5 and activates TAA1 expression in vivo. A, A cartoon showing regions amplified in the ChIP-qPCR experiment. B, Expression of endogenous TAA1 is induced in heat-treated HSP_pro:ARR1C-3FL seedlings (FL, FLAG tag). C, ChIP-qPCR assays showing that P1 and P5 regions were specifically enriched as the ARR1C binding regions. Representative data from three biological replicates were shown. D and E, Protoplast transient expression assay showing ARR1C activates the expression of the LUC through its specific interaction with the ARR binding sites on P1/P5. Error bars represent se of mean (sem; n = 3). **Significant difference to corresponding control with P < 0.01.

Figure 4.

ARR12C directly interacts with ARR1C to promote its transcriptional activity on TAA1. A, BiFC showing interaction between ARR12C and ARR1C. Scale bar, 25μm. B and C, Protoplast transient expression assay showing ARR12C promotes ARR1C-activated expression of LUC reporter gene driven by P1 (B) or P5 (C). Error bars represent se of mean (sem; n = 3). **Significant difference to corresponding control with P < 0.01.

Figure 5.

The relationship between cytokinin and TAA1. A, 6-BA alters TAA1 expression pattern in an ARR-dependent manner. B, 6-BA enhances TAA1 expression in cotyledons and true leaves in an ARR-dependent manner. Error bars represent sem (n = 3). C, Response to 6-BA in roots requires functional TAA1. Regions between arrowheads are primary roots. Quantitative measurement of root length is shown in the right. Error bars represent sem (n ≥ 15). * and **Significant difference to Col-0 with P < 0.01 and 0.001, respectively (Student’s t test).

Figure 6.

Exogenous cytokinin enhances auxin signaling in a TAA1-dependent manner. Expression pattern of DR5:GUS in the wild type or sav3-1/taa1 background.

Figure 7.

arr mutants display phenotypes consistent with having reduced auxin level. A, Germination rates of freshly harvested seeds with (right) or without (left) imbibition (n = 3, ≥70 seeds/sample). B, Hypocotyl length of Col-0, sav3-1, and arr1,10,12 seedlings grown in Wc or shade (n ≥ 15). C, arr1,12 is hypersensitive to NPA in shade (n ≥ 15). D, Quantification of the free IAA levels in 5-d-old light-grown (Wc) Col-0 and arr1,10,12 seedlings treated with Wc or shade for 2 h. E, Quantification of the free IAA levels in 2-d-old dark-grown Col-0 and arr1,10,12 seedlings. Error bars represent sem. * and **Significant difference to controls with P < 0.05 and 0.01, respectively (Student’s t test).

Figure 8.

ARRs are required for transcriptional regulation of TAA1 in planta. A, Expression of TAA1 in Col-0, scr-1, and arr1-3 scr-1 roots (n = 3). B, Root phenotypes of 7-d-old light-grown seedlings. Regions between arrowheads are primary roots. Quantitative measurements of the root length are shown at the bottom (n ≥ 15). Error bars represent sem. * and **Significant difference between samples with P < 0.05 and 0.01, respectively (Student’s t test).

Figure 9.

Transcriptional regulation of TAA1 by light requires ARRs. A, Expression of TAA1 is transiently induced by light. Two-day-old dark-grown (D) seedlings were either kept in dark for 1 day or exposed to light (L) for 12 or 24 h. B, Light-induced changes in GUS expression pattern of TAA1_pro:TAA1g-GUS in wild type or arr1,12 mutant background. Scale bar, 1 mm. C, Cotyledon opening rate of 2-d-old dark-grown seedlings transferred to light for the indicated amount of time (n ≥ 60). D, Cotyledon areas of 2-d-old dark-grown seedlings transferred to light for the indicated amount of time (n = 30). E, Expression of TAA1, SAUR14, SAUR50, and SAUR65 in 2-d-old dark (D)-grown seedlings exposed to light (L) for 0, 2, and 4 h. Error bars represent sem. A, C, D, and E, * and **Significant difference to the corresponding controls with P < 0.05 and 0.01 (Student’s t test), respectively.

Figure 10.

DELLA protein GAI promotes the transcriptional activation of TAA1 by ARR1C in cotyledons. A and B, Protoplast transient expression assay showing GAI promotes ARR1C-activated expression of LUC reporter gene driven by P1 (A) or P5 (B). C, PAC treatment enhanced GUS expression in cotyledons of dark-grown P1-CORE_pro:GUS seedlings. Scale bar, 1 mm. D, GUS expression pattern of TAA1_pro:TAA1g-GUS in wild type or arr1,12 mutant background in response to PAC treatment. Scale bar, 1 mm. E, qRT-PCR results showing TAA1 expression in cotyledons in response to PAC. For experiments in C to E, seedlings were grown in dark on 1/2 MS medium supplemented with or without 0.5 μm of PAC for 2 d (2D). Error bars represent se of mean (sem; n = 3). **Significant difference between the two samples with P < 0.01 (Student’s t test).

Figure 11.

EIN3 directly interacts with ARR1 to promote its transcriptional activity on TAA1. A, Ethylene response of Col-0, sav3-1,arr1, and arr1,12 roots. B, BiFC showing interactions between ARR1C and EIN3. Scale bar, 25 μm. C and D, Protoplast transient expression assay showing EIN3 promotes ARR1C-activated expression of LUC reporter gene driven by P1 (C) or P5 (D). E, A proposed model showing that the type B ARRs function as central transcriptional regulators of TAA1. The type B ARRs can directly bind to the cis elements on P1 and/or P5 to promote TAA1 expression. N terminus of the type B ARRs inhibits the transcriptional activity of the C terminus, which can be relieved by the activation of the cytokinin pathway. Developmental and environmental signals may regulate TAA1 expression through modulating cytokinin metabolism or signaling. Alternatively, they may regulate transcription factors such as DELLAs and EIN3 that directly interact with the type B ARRs and modulate their transcriptional activities. Arrows with dashed lines indicate proposed interactions. Error bars represent se of mean (sem; n = 3). **Significant difference to the corresponding controls with P < 0.01 (Student’s t test).