Identification of a novel jasmonate-responsive cis-element and functional analysis of trans-acting factor, AtBBD1

Abstract

Jasmonates (JAs) are important regulators of plant biotic and abiotic stress responses and development. AtJMT in Arabidopsis thaliana and BcNTR1 in Brassica campestris encode jasmonic acid carboxyl methyltransferases, which catalyze methyl jasmonate (MeJA) biosynthesis and are involved in JA signaling. Their expression is induced by MeJA application. To find out JA-responosive elements in AtJMT and BcNTR1 promoters, various promoter deletion fragment of each promoter were recombined with GUS reporter gene. Each promoter deletion construct was introduced into Arabidopsis plant. Each transgenic plant was treated with MeJA and transcript of reporter gene was checked to test JA-responsive gene induction. It was revealed that specific region of AtJMT (-2,294~-2,280) and BcNTR1 (-3,518~-3,480) promoter was important for JA-responsive gene expression. Alignment of these sequences showed that completely same 6 nucleotides (TCCTGA) were found in both promoter regions. It was expected that this element was critical for JA-responsive gene expression. To confirm the function of this element, multimeric construct of this element was made and introduced into Arabidopsis. RT-PCR and GUS staining of transgenic plant after MeJA treatment were showed that this 6 nucleotide (TCCTGA) was important for JA-responsive gene expression. Therefore, this element was defined as a novel JA-responsive cis-element (JARE) in the AtJMT and BcNTR1 promoters, because the JARE is distinct from other JA-responsive cis-elements previously reported. On the other hand, Y1H screening was also carried out to identify a trans-acting factor. JA-responsive region (-3,518~-3,390) of BcNTR1 promoter was used as a bait and MeJA treated Arabidopsis cDNA library was used for Y1H screening. As a result, AtBBD1, an Arabidopsis homologue of the Oryza minuta bifunctional nuclease in basal defense response (OmBBD1), was screened out. To find out specific cis-element which AtBBD1 bind to, we divided and mutated the bait fragment for Y1H assay. Y1H assay results showed that AtBBD1 bound to the JARE and it extended JARE to (G/C)TCCTGA. To investigate the function of AtBBD1 in JA signaling pathway, Y2H assay was carried out between AtBBD1 and AtJAZs proteins. It was found that AtJAZ1 and 4 interact with AtBBD1. In vitro pull down assay also showed that AtBBD1 and AtJAZ1 was interact with each other. Protein deletion experiment with Y2H showed that N-terminal region containing HCR motif of AtBBD1 was interacted with TIFY containing region of AtJAZ1. To investigate the function of AtBBD1 on its target gene and JA signaling pathway, knockout (atbbd1) and overexpression plants were analysed. Single knockout of AtBBD1 showed no significant difference on AtJMT gene expression in response to MeJA treatment. But double knockout (atbbd1 atbbd2) plant of AtBBD1 and AtBBD2, which has 81% amino acid sequence similarity, showed that AtJMT expressed higher than wild-type in response to MeJA. But JR2, JA-responsive marker gene, expression was reduced in atbbd1 atbbd2. On the other hand, AtBBD1 overexpressing plant showed that AtJMT expression was reduced and JR2 expression was enhanced in response to MeJA treatment. Therefore, AtBBD1 and its close homologue AtBBD2 are functionally redundant and act as negative regulators of AtJMT expression. However, AtBBD1 positively regulated the JA-responsive expression of JR2. To understand AtBBD1 repression mechanism, chromatin acetylation level was tested by Chromatin immunoprecipitation (ChIP) with specific acetylated antibody because it was known that AtJAZ1 interacted with histone deacetylase directly or indirectly previouly. ChIP data from knockout and overexpression plants revealed that repression of AtJMT is associated with reduced histone acetylation in the promoter region containing the JARE. These results show that AtBBD1 interacts with JAZ proteins, binds to the JARE and represses AtJMT expression.