A Novel Plant Cysteine Protease Has a Dual Function as a Regulator of 1-Aminocyclopropane-1-Carboxylic Acid Synthase Gene Expression

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.

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.

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.

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.

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.