A novel plant cysteine protease has a dual function as a regulator of 1-aminocyclopropane-1-carboxylic Acid synthase gene expression

Abstract

The hormone ethylene influences plant growth, development, and some defense responses. The fungal elicitor Ethylene-Inducing Xylanase (EIX) elicits ethylene biosynthesis in tomato (Lycopersicon esculentum) and tobacco (Nicotiana tabacum) leaves by induction of 1-aminocyclopropane-1-caboxylic acid synthase (Acs) gene expression. A minimal promoter element in the LeAcs2 gene required for EIX responsiveness was defined by deletion analysis in transgenic tomato plants. The sequence between -715 and -675 of the tomato Acs2 gene was found to be essential for induction by EIX. A Cys protease (LeCp) was isolated that specifically binds to this cis element in vitro. Ectopic expression of LeCp in tomato leaves induced the expression of Acs2. Moreover, chromatin immunoprecipitation showed that LeCp binds in vivo to the Acs promoter. We propose a mechanism for the dual function of the LeCp protein. The protease acts enzymatically in the cytoplasm. Then, upon signaling, a small ubiquitin-related modifier protein binds to it, enabling entrance into the nucleus, where it acts as a transcription factor. Thus, LeCp can be considered a dual-function protein, having enzymatic activity and, upon elicitor signaling, exhibiting transcriptional factor activity that induces LeAcs2 expression.

Figure 1.

Induction of GUS Activity in Transgenic Tomato Harboring the Deleted Acs2 Promoter. (A) Scheme of the different promoter deletions. The 3058-bp promoter and the deleted promoter fragments were cloned upstream of the uidA reporter gene (GUS). (B) Transgenic plants harboring the different promoter deletions as indicated were treated for 4 h with EIX (1 μg/mL) elicitor. The GUS induction ratio (treated/untreated) is shown. The data in the graph correspond to the mean ± se of three independent experiments.

Figure 2.

In Vitro Binding of Nuclear Proteins to −715 to −675 of the Acs2 Promoter. Gel-shift binding assay with nuclear proteins extracted from EIX-treated tobacco plants that were incubated with 30 fmol of end-labeled 40-bp fragment (corresponding to −715 to −675 in the Acs2 promoter) in the presence of 2 μg of poly(dI-dC) and 10-, 30-, or 100-fold excess competitor fragments, as indicated. After the binding step, the DNA–protein complexes were fractionated by PAGE and detected by autoradiography. The bound complexes are identified at left.

Figure 3.

Similarity of LeCp to Cys Proteases. Alignment of the LeCp amino acid sequence (accession number AJ841791) with those of Cys proteases from Nicotiana tabacum (accession number AB075947), Citrus sinensis (accession number P49043), Arabidopsis thaliana (accession number Q39119), Ipomoea batatas (accession number AAF69014.1), Canavalia ensiformis (accession number P49046), and Homo sapiens legumain (accession number Q99538). The bestfit and pretty box programs (GCG sequence analysis software package, version 10.0; Accelrys, San Diego, CA) were used to create the best alignment. Identical amino acids are shaded in black, and conservative substitutions are shaded in gray. The triangle marks the conserved Cys that is essential for the enzymatic activity. A circle marks the SUMOlation sites.

Figure 4.

In Vitro Binding of LeCp to Promoter Sequences of Acs2. Gel-shift binding assay was performed by incubating LeCp (0.1 μg) with 30 fmol of end-labeled 40-bp fragment (corresponding to −715 to −675 in the Acs2 promoter) and 2-, 10-, 25-, 50-, or 250-fold excess competitor fragments, as indicated (left). LeCp (0.1 μg) was also incubated with 30 fmol of end-labeled wild-type (W.T.) or mutated 40-bp fragment (corresponding to −715 to −675 in the Acs2 promoter), as indicated, in the presence of 2 μg of poly(dI-dC) (right). After the binding step, the DNA–protein complexes were fractionated by PAGE and detected by autoradiography. Bound complexes are identified at left. oligo, oligonucleotide.

Figure 5.

In Vivo Induction of the GUS Reporter Gene by LeCp Expression. Transgenic L. esculentum cv VF36 plants harboring the 3058-bp promoter and the deleted promoter fragments (as indicated) cloned upstream of the uidA gene were infiltrated with Agrobacterium GV3101 (OD₆₀₀ = 0.1) containing either the Pro_35S:LeCp construct or empty vector (control) (A), infected with TRV, TRV-tPDS, or TRV-LeCp followed by infection with Agrobacterium GV3101 (OD₆₀₀ = 0.1) containing the Pro_35S:tvEix construct (B), or infiltrated with Agrobacterium GV3101 (OD₆₀₀ = 0.1) containing the mutated Pro_35S:LeCp construct (C). GUS activity was monitored at 48 h after injection.

Figure 6.

In Vivo Binding of LeCp Protein to Promoter Sequences of the Acs Gene. Transgenic tobacco plants harboring Pro_35S:LeCp-GFP were subjected to ChIP with anti-GFP antibodies. PCR was performed using primers corresponding to sequences located near the 40-bp motif in the Acs2 promoter and primers corresponding to the actin gene. As a control for DNA input into the ChIP assays, aliquots of sonicated chromatin were analyzed by PCR using the same primers as those used in the ChIP assays.

Figure 7.

Interaction of LeCp with T-SUMO. (A) EGY48 yeast cells containing T-SUMO (in pEG202) and LeCp (in pJG4-5) (row 1), T-SUMO (in pEG202) and mutated LeCp (in pJG4-5) (row 2), Bicoid (in pEG202) and LeCp (in pJG4-5) (row 3), empty vector (in pEG202) and LeCp (in pJG4-5) (row 4), or T-SUMO (in pEG202) and Rubisco (in pJG4-5) (row 5) were grown on galactose medium lacking the amino acids His, Ura, and Trp and supplied with X-Gal. (B) Transgenic L. esculentum cv VF36 plants harboring the 3058-bp promoter cloned upstream of the uidA gene were infiltrated with Agrobacterium GV3101 (OD₆₀₀ = 0.1) containing either the Pro_35S:LeCp construct or the SUMOlation signal–mutated Pro_35S:LeCp construct. GUS activity was monitored at 48 h after injection.