NtCP56, a new cysteine protease in Nicotiana tabacum L., involved in pollen grain development

Abstract

Proteinases play a critical role in developmental homeostasis and in response to environ-mental stimuli. Our present research reports that a new cysteine protease, NtCP56, is involved in the development of pollen grains in Nicotiana tabacum L. The NtCP56 gene, which encodes a protein of 361 amino acid residues with a calculated molecular mass of 40 kDa, is strongly expressed in anthers. The recombinant NtCP56 showed a high activity towards casein. Kinetic analysis revealed a Km of 2.20 mg ml−1 and Vmax of 11.07 μg ml−1 min−1. The recombinant NtCP56 retained more than 50% of its maximum enzymatic activity from 20 °C to 60 °C with an optimum Tm range of 30–50 °C. The enzyme had a maximum activity at approximately pH 6.5. Suppression of the NtCP56 gene in anti-sense transgenic tobaccos resulted in the sterility of pollen grains. Our data indicated that, as a cysteine protease, NtCP56 might play an important role in pollen development.

Isolation of cDNA from Jacaratia mexicana encoding a mexicain-like cysteine protease gene

“…CP gene identification has occurred in several plant sources, such as Mesembryanthemum crystallinum (Forsthoefel et al, 1998), Daucus carota (Mitsuhashi et al, 2004), and C. candamarcensis (Pereira et al, 2001). Over-expression of CP genes from plant sources, like C. papaya (Taylor et al, 1999), Ipomoea batatas (Chen et al, 2009;2010), Nicotiana tabacum (Beyene et al, 2006;Zhang et al, 2009), and C. candamarcensis (Corrêa et al, 2011), has also been reported and shown to be encouraging examples of the aims presented in this study. The main objective of this research was to identify the gene sequences coding for the proteases that comprise the latex of J. mexicana.…”

The Cysteine Protease CEP1, a Key Executor Involved in Tapetal Programmed Cell Death, Regulates Pollen Development in Arabidopsis

“…The expression, extraction, purification, and renaturation of the CEP1 protein were performed according to the procedure described by Zhang et al (2009).…”

“…The expression, extraction, purification, and renaturation of the CEP1 peptide were performed according to the procedures described by Zhang et al (2009).The purified recombinant peptide (5 mg) was used to immunize rabbits at 3-week intervals. The specificity of the CEP1 antibody was confirmed by hybridization with a membrane blotted with protein extracts from the buds of Arabidopsis (Supplemental Figure 7).…”

Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination

“…All of the C1A proteases contain several disulfide bonds and share three conserved catalytic residues (Cys, His, and Asn) in the catalytic triad, a trait that is common to all Cysproteases, and they also contain a Gln involved in maintaining an active enzyme conformation. In addition, the GCNGG motif has been identified in all C1A protease members, and the noncontiguous ERFNIN motif has been found in cathepsin L-and H-like proteins (Grudkowska and Zagdanska, 2004;Zhang et al, 2009). C1A Cys-proteases from plants are synthesized as preproteins, processed either automatically or with the aid of processing enzymes, transported via the trans-Golgi network, and finally either are stored in the vacuoles and lysosomes or externally secreted (Grudkowska and Zagdanska, 2004).…”