Abstract
Brassinosteroids (BRs) are a group of major phytohormones playing critical roles in plant growth and development. Within the last two decades, key events of BR biosynthesis and signal transduction have been gradually elucidated. The detailed molecular mechanisms controlling bioactive levels of BRs, however, are not fully understood. TCP1 is a member of class ii TCP proteins in Arabidopsis thaliana. The role of TCP1 in BR biosynthesis was discovered by an activation tagging analysis aiming to screen for genetic suppressors of an intermediate allele named bri1-5 of the BR receptor gene BRI1. Overexpression of TCP1 partially suppresses the defective phenotypes of bri1-5 via direct upregulation of DWF4, one of the target genes of TCP1.
Key words: Arabidopsis thaliana, brassinosteroids, DWF4, TCP1, activation tagging
Previous studies using pea plants indicated that BRs cannot be transported in a long distance way as other phytohormones do.1 BR homeostasis, therefore, must be precisely regulated within a plant cell. Both excessive and deficient amount of BRs are inadequate to optimal plant growth and development. Generally speaking, bioactive levels of BRs are mainly controlled by three different mechanisms, positive regulation, negative feedback regulation and catabolism. Positive/negative feedback regulation controls the speed of biosynthesis, whereas catabolism mediates the rate of degrading excessive amount of BRs. It was found that the expression levels of a number of crucial BR biosynthetic genes are controlled by negative feedback mechanisms.2–8 For example, when endogenous BRs are depleted by a specific BR biosynthetic inhibitor, brassinazole (BRZ),9 at least five known BR biosynthetic genes including DET2, DWF4, CPD, BR6ox1 and ROT3 are upregulated.6 Whereas, four of these five genes (DWF4, CPD, BR6ox1 and ROT3) were found to be downregulated upon the supplementation of exogenous BL, the final product of BR biosynthetic pathway and the most active BR.6 It is now clear that the negative feedback response of BRs is through two dual-role transcription factors, BZR1 and BES1.3,7 Each of the aforementioned five BR biosynthesis gene promoters contains at least one specific binding site for either BZR1 or BES1 or both.10,11 BZR1 and BES1 act either as repressors or activators for thousands of downstream BR response genes.8 Within the past decade, a number of proteins involved in BR catabolism have also been identified. For example, BNST3 from Brassica napus was found to inactivate BRs by sulfonation.12 Arabidopsis BAS1, SHK1/SOB7/CHI2, UGT73C5, BEN1 are thought to be involved in inactivating BRs with distinctive mechanisms.13–18 Positive regulation of BR biosynthesis had been less understood until TCP1 and CESTA were identified as transcription factors directly stimulating the transcription of DWF4 and CPD, respectively.19,20 In this addendum, we discuss the information of TCP1 in regulating BR biosynthesis by upregulating the expression of a key BR biosynthesis gene, DWF4.
Both dwf4-1D and tcp1-1D were Identified as Dominant Genetic Suppressors of bri1-5
In higher plants, most of the genes contain at least one additional copy, which makes reverse genetics a less powerful strategy to reveal the biological roles of these genes. To overcome this problem, we employed a gain-of-function approach called activation tagging to identify novel genes regulating BR homeostasis and signal transduction via screening for suppressers of bri1-5, an intermediate BR receptor mutant. From a large scale activation tagging transgenic pool, we identified dwf4-1D and tcp1-1D as two authentic genetic suppressors of bri1-5.19 Overexpression of DWF4 and TCP1 can partially suppress the defective phenotypes of bri1-5 including the flowering time, petiole length, rosette width and plant height (Fig. 1). Overexpression of TCP1 also suppresses biosynthetic mutant det2 but cannot suppress null alleles of BRI1, indicating that TCP1 is indeed involved in BR signal transduction or biosynthesis. Because there lacks the T-DNA insertion lines for TCP1 in the Arabidopsis Biological Resource Center (ABRC), we constructed a chimeric repressor mutant tcp1-SRDX. Overexperession of tcp1-SRDX resulted in a typical dominant negative phenotype which is similar to the BR deficient or signaling mutants such as det2 or bri1-5. These results further suggested that TCP1 has a role in a BR related pathway.
Figure 1.
Phenotypes of bri1-5 and bri1-5 tcp1-1D. The plants were photographed five weeks after germination.
TCP1 Positively Regulate BR Biosynthesis via its Direct Regulation of the Transcription of DWF4
Interestingly, the dwarfed tcp1-SRDX transgenic plants can be rescued by exogenously supplied BL, but not by any other tested major plant hormones such as auxins, gibberellins or cytokinins, hinting that dwarfed tcp1-SRDX plants may have resulted from the disruption of BR biosynthesis rather than BR signal transduction. Because TCP proteins usually act as transcription factors, we proposed that TCP1 may directly regulate the expression of a gene/genes involved in BR biosynthesis. To discover the true TCP1 target gene, we compared the amount of BR biosynthetic intermediates in WS2, tcp1-1D and tcp1-SRDX plants to examine which specific BR biosynthetic step has been altered. Our results showed that the catalytic capability of DWF4 is obviously upregulated in tcp1-1D and downregulated in tcp1-SRDX plants. ChIP analysis indicated that TCP1 can interact with the promoter region of DWF4. Electrophoretic mobility shift assays (EMSA) solidified the hypothesis that TCP1 directly regulate the transcription of DWF4 via its association with its binding site within the DWF4 promoter (unpublished data). The facts that tcp1-1D cannot suppress dwf4 and the identification of dwf4-1D as one of the genetic suppressors of bri1-5 are consistent with the hypothesis that TCP1 is a positive regulator mediating the transcription of the key BR biosynthetic gene DWF4.
Perspectives
Regarding the BR homeostasis, positive regulation of BR biosynthesis is the least understood research area in the field. Identification of TCP1 in regulating DWF4 transcription and CESTA in mediating CPD expression opens up a novel avenue in BR homeostasis research.19,20 In the future, the true TCP1 binding sequence in DWF4 promoter has to be confirmed by a series of EMSA analyses. In addition, how TCP1 expression can respond various endogenous and exogenous cues will be another attractive focus to understand how BR biosynthesis is stimulated by internal and environmental signals. We are also testing all 23 TCP1 homolgs in Arabidopsis genome to determine all possible functional redundant genes of TCP1. Once determined, we will further use reverse genetic tools to test their significance in regulating plant growth and development.
Acknowledgments
The authors' research group is currently supported by National Natural Science Foundation of China Grants 90917019 (to J.L.), National Basic Research Program of China Grant 2011CB915401 (to J.L.) and 31070283 (to X.G.)
Abbreviations
- TCP
TEOSINTE BRANCHED 1, CYCLOIDEA, and PCF
- DWF4
DWARF4
- BRs
brassinosteroids
- CPD
CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM
- BR6ox1
BR-6-oxidase 1
- DET2
DE-ETIOLATED 2
- ROT3
ROUNDFOLIA 3
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