Cloning and characterization of a gibberellin-induced RNase expressed in barley aleurone cells

Abstract

We cloned a cDNA for a gibberellin-induced ribonuclease (RNase) expressed in barley (Hordeum vulgare) aleurone and the gene for a second barley RNase expressed in leaf tissue. The protein encoded by the cDNA is unique among RNases described to date in that it contains a novel 23-amino acid insert between the C2 and C3 conserved sequences. Expression of the recombinant protein in tobacco (Nicotiana tabacum) suspension-cultured protoplasts gave an active RNase of the expected size, confirming the enzymatic activity of the protein. Analyses of hormone regulation of expression of mRNA for the aleurone RNase revealed that, like the pattern for alpha-amylase, mRNA levels increased in the presence of gibberellic acid, and its antagonist abscisic acid prevented this effect. Quantitative studies at early times demonstrated that cycloheximide treatment of aleurone layers increased mRNA levels 4-fold, whereas a combination of gibberellin plus cycloheximide treatment was required to increase alpha-amylase mRNA levels to the same extent. These results are consistent with loss of repression as an initial effect of gibberellic acid on transcription of those genes, although the regulatory pathways for the two genes may differ.

Figure 1

Sequence analysis. A, A comparison is presented of predicted amino acid sequences of barley aleurone RNase (GAR-RNase) (accession no. AF000939) and the leaf RNase (Leaf) encoded by the gene isolated from a barley genomic library (accession no. AF000940) and known sequences from the database for RNases of zinnia (accession no. U19924; Zinnia) and Arabidopsis (accession no. U05206; At). Numbering begins with the initial Met. Asterisks indicate active-site His residues, and conserved Glu is shown in bold. Five highly conserved regions are underlined and labeled C1 to C5. A 23-amino acid insert unique to GAR-RNase is present from residues 73 to 95. Dots above residues indicate a region that is predicted to form an α-helix. Vertical lines show positions of predicted signal peptide cleavage. B, Kyte-Doolittle hydropathy plot of the predicted protein sequence of GAR-RNase cDNA (Kyte and Doolittle, 1982). The area highlighted in gray is the 23-amino acid insert unique to GAR-RNase. Numbering of amino acids is the same as in A. S, Hydrophobic region corresponding to signal peptide.

Figure 2

Southern blot probed with GAR-RNase cDNA. DNA from barley cvs Morex (lanes M) and Steptoe (lanes S) was digested with EcoRI (RI; lanes 1 and 2), EcoRV (RV; lanes 3 and 4), or HindIII (H; lanes 5 and 6), electrophoresed, transferred to a membrane, and hybridized with the GAR-RNase cDNA probe. Sizes of molecular mass markers (in kb) are indicated to the left.

Figure 3

Expression of recombinant GAR-RNase in tobacco suspension-cultured protoplasts. A, Coomassie blue-stained gel from SDS-PAGE. B, RNase activity gel. Lanes M, Molecular mass markers with size in kD to the right. Lanes 1, Cell extract from protoplasts expressing GAR-RNase; lanes 2, extract from protoplasts expressing E. coli GUS; lanes 3, medium from GAR-RNase-expressing cells; and lanes 4, medium from control cells. Dots indicate approximately 18-kD RNase activity in both cell extracts, arrow indicates approximately 26-kD RNase activity in extract from GAR-RNase-expressing cells, and asterisk indicates approximately 26-kD RNase activity in medium from GAR-RNase-expressing cells.

Figure 4

Hormone effects on mRNA expression. A, Hybridization with the GAR-RNase probe. Total RNA was isolated from barley aleurone layers treated for 24 h with no hormone (0; lane 1), with GA₃ (G; lane 2), or with GA plus ABA (G + A, lane 3). Following electrophoresis, RNA was transferred to nitrocellulose and blots were probed sequentially. First, the blot was probed with the full-length GAR-RNase cDNA. After the signal was collected by phosphor imager analysis, the blot was stripped and probed with a mixture of high-pI α-amylase cDNA (Amy) and PAPI probes. Sizes (in kb) are indicated to the right of each panel. B, Hybridization with barley leaf RNase probe. Total RNA blots were prepared and probed as for Figure 3A, except that here the RNase probe was an approximately 600-bp SalI fragment of coding sequence from the RNase gene. Lane 1, No hormone (0); lane 2, 24 h with GA₃ (G); and lane 3, leaf RNA (L). Sizes of mRNA (in kb) are indicated to the right of each panel.

Figure 5

Effect of Chx on mRNA expression patterns. A blot carrying 20 μg/lane of total aleurone RNA from de-embryonated half-seeds not treated (0; lanes 1, 5, and 9), treated with 50 μm Chx (C; lanes 2, 6, and 10), treated with 10⁻⁶ m GA (G; lanes 3, 7, and 11), or treated with GA plus Chx (GC; lanes 4, 8, and 12) for 6 h was probed sequentially with cDNAs for GAR-RNase, PAPI, and high-pI α-amylase (Amy). After the blot was washed at high stringency, images were captured and quantitated with a phosphor imager. The signals for PAPI in each lane were used to correct for differences in loading. RA, Relative abundance, where the value obtained with GA was set to 1.0.