Alternative splicing of basic chitinase gene PR3b in the low-nicotine mutants of Nicotiana tabacum L. cv. Burley 21

Abstract

Two unlinked semi-dominant loci, A (NIC1) and B (NIC2), control nicotine and related alkaloid biosynthesis in Burley tobaccos. Mutations in either or both loci (nic1 and nic2) lead to low nicotine phenotypes with altered environmental stress responses. Here we show that the transcripts derived from the pathogenesis-related (PR) protein gene PR3b are alternatively spliced to a greater extent in the nic1 and nic2 mutants of Burley 21 tobacco and the nic1nic2 double mutant. The alternative splicing results in a deletion of 65 nucleotides and introduces a premature stop codon into the coding region of PR3b that leads to a significant reduction of PR3b specific chitinase activity. Assays of PR3b splicing in F₂ individuals derived from crosses between nic1 and nic2 mutants and wild-type plants showed that the splicing phenotype is controlled by the NIC1 and NIC2 loci, even though NIC1 and NIC2 are unlinked loci. Moreover, the transcriptional analyses showed that the splicing patterns of PR3b in the low-nicotine mutants were differentially regulated by jasmonate (JA) and ethylene (ET). These data suggest that the NIC1 and NIC2 loci display differential roles in regulating the alternative splicing of PR3b in Burley 21. The findings in this study have provided valuable information for extending our understanding of the broader effects of the low-nicotine mutants of Burley 21 and the mechanism by which JA and ET signalling pathways post-transcriptionally regulate the activity of PR3b protein.

Keywords: PR3b; Alternative splicing; ethylene; jasmonate; low-nicotine mutant; tobacco..

Fig. 1.

Induced expression patterns of the nicotine synthetic gene PMT1 and PR protein genes in the roots of wild-type tobacco Burley 21. (A) Expression pattern assay by semi-quantitative RT-PCR. The representative results of three independent replicates are shown. The sizes of the amplified products are indicated on the right. (B) Transcript levels of PMT1 and PR protein genes based on qRT-PCR analysis. Ctrl indicates untreated control. JA and ACC indicate treatment with JA and ACC, respectively. The transcription level of each gene in the Ctrl is set as ‘1’. Actin was used as an internal control for both semi-quantitative RT-PCR and qRT-PCR.

Fig. 2.

Transcription patterns of PMT1 and PR protein genes in the wild type and in low-nicotine mutants of Burley 21. (A) Expression patterns of PMT1 and PR protein genes in roots by semi-quantitative RT-PCR assay. (B) Expression levels of PMT1 and PR protein genes determined by qRT-PCR. The transcription level of each gene in the Ctrl is set as ‘1’. (C) Transcripts of PR3b gene in leaves. (A, C) The representative results of three independent replicates and the sizes of the amplified products are indicated on the right. WT indicates wild type Burley 21. nic1, and nic2 indicate low-nicotine mutant alleles of NIC1 and NIC2, and nic1nic2 indicates the double mutant. The asterisk indicates primer dimers in the amplification products of the PR3b gene. Actin was used as an internal control.

Fig. 3.

Characterization of the alternative splicing of PR3b. (A) Schematic gene structure of PR3b. The details are shown in Supplementary Fig. S1. (B) Alignment of the alternatively spliced PR3b fragment (Spliced) against the native PR3b cDNA (sequence with labelled nucleotide numbers). The numbers indicate the position of the indicated nucleotide at the native PR3b coding region. The alternative spliced region is highlighted by rectangles and dashes between the rectangles indicate the excised region by the alternative splicing. Positions corresponding to the single-nucleotide mutation sites are indicated by red characters. The amino acid sequences in green are deduced from the native PR3b cDNA. The amino acid sequence highlighted by the grey bar shows the coding change region caused by alternatively splicing of PR3b; the dash in the amino acid sequence indicates the position of the stop codon. (C) Comparison of RT-PCR and genomic DNA PCR (gDNA-PCR) products amplified using the same primers. Marker indicates the DNA molecular marker. WT, wild type; nic1, low-nicotine mutant nic1.

Fig. 4.

Specific chitinase activity of native and alternatively spliced PR3b. (A) Conserved α-helixes (blue arrows), β-sheets (pink bars), and amino acids (red characters) of PR3b compared with the catalytic cores of GH18 family chitinases (Hurtado-Guerrero and van Aalten, 2007). The region affected by alternative splicing is underlined with a black line. (B) Purified proteins of GST-tagged PR3b and GST separated by SDS-PAGE gel. ‘GST-PR3b’ is the GST-tagged protein of native PR3b; ‘GST-PR3b Spliced’ is the GST-tagged protein of spliced PR3b. Marker is the protein standard ruler. The picture shown is a combination of representative lanes from gels with serial elutions of the purified proteins. (C) Enzyme-specific activity of GST-tagged native and alternatively spliced PR3b. One unit equals 1 μmol of released 4-MU. GST is used for the control reaction. Values are the average of three replicates. Error bar, mean ±SD. The asterisk indicates a significant difference between the paired data sets (P <0.05, Student’s t test).

Fig. 5.

Genetic linkage between alternative splicing of PR3b and the NIC1 locus in the Burley 21 background. (A) Alternative splicing of PR3b in individual F₂ plants of a cross between nic1 and wild-type Burley 21. (B) Transcript levels of PMT1 in the roots of individual F₂ plants. Transcript level of PMT1 in the roots of wild-type Burley 21 was set as ’1’. Actin was used as an internal control. (C) Leaf nicotine content of individual F₂ plants. The values shown are the means of three technical replicates. Error bar, mean ±SD.

Fig. 6.

Phytohormone-induced splicing patterns of PR3b. (A) Splicing patterns of PR3b in the wild type (WT) and in low-nicotine mutants (nic1, nic2, and nic1nic2). The representative results of three independent replicates are shown. The asterisk indicates the primer dimers in the PCR amplification products. (B) Quantification of specific transcript of native PR3b. (C) Quantification of specific transcript of spliced PR3b. Ctrl indicates the untreated control; JA, ACC, and JA+ACC (the combination of JA and ACC) indicate different phytohormone treatments. The PR3b transcript level in the WT of the Ctrl treatment is set as ‘1’ (B, C). Actin was used as an internal control.