Two alternative splicing variants of a wheat gene TaNAK1, TaNAK1.1 and TaNAK1.2, differentially regulate flowering time and plant architecture leading to differences in seed yield of transgenic Arabidopsis

Abstract

In wheat production, appropriate flowering time and ideal plant architecture are the prerequisites for high grain yield. Alternative splicing (AS) is a vital process that regulates gene expression at the post-transcriptional level, and AS events in wheat have been found to be closely related to grain-related traits and abiotic stress tolerance. However, AS events and their biological roles in regulating flowering time and plant architecture in wheat remain unclear. In this study, we report that TaNAK1 undergoes AS, producing three splicing variants. Molecular characterization of TaNAK1 and its splicing variants demonstrated that all three protein isoforms have a conserved NB-ARC domain and a protein kinase domain, but the positions of these two domains and the length of the protein kinase domains are different among them, implying that they may have different three-dimensional structures and therefore have different functions. Further investigations showed that the two splicing variants of TaNAK1, TaNAK1.1 and TaNAK1.2, exhibited different expression patterns during wheat growth and development, while the other one, TaNAK1.3, was not detected. Subcellular localization demonstrated that TaNAK1.1 was mainly localized in the cytoplasm, while TaNAK1.2 was localized in the nucleus and cytoplasm. Both TaNAK1.1 and TaNAK1.2 exhibit protein kinase activity in vitro. Ectopic expression of TaNAK1.1 and TaNAK1.2 in Arabidopsis demonstrated that these two splicing variants play opposite roles in regulating flowering time and plant architecture, resulting in different seed yields. TaNAK1.2 positive regulates the transition from vegetative to reproductive growth, plant height, branching number, seed size, and seed yield of Arabidopsis, while TaNAK1.1 negatively regulates these traits. Our findings provide new gene resource for regulating flowering time and plant architecture in crop breeding for high grain yield.

Keywords: TaNAK1; alternative splicing; flowering time; plant architecture; seed yield; wheat.

Figure 1

TaRK4B-1 is subject to alternative splicing. (A) Gene structure of three alternative splicing variants TaRK4B-1.1, TaRK4B-1.2 and TaRK4B-1.3. CS, constitutive splicing; ES, exon skipping; VFE, variable first exon; Alt 3’ ss, alternative 3’ splice site. (B) Schematic representation of the three TaRK4B-1 isoforms; Pkinase, protein kinase domain; NB-ARC, NB-ARC domain. (C) Amino acid sequence alignment of the protein kinase domains in the three TaRK4B-1 isoforms.

Figure 2

Expression patterns of TaNAK1 splicing variants and subcellular localization and the autophosphorylation activities of two protein isoforms TaNAK1.1 and TaNAK1.2. (A) The spatiotemporal expression profiles of the splicing variants of TaNAK1 in wheat detected by semi-quantitative reverse transcriptase-polymerase chain reaction with Actin (TraesCS1A01G020500) as a reference gene. TaNAK1.1 and TaNAK1.2, two splicing variants of TaNAK1; Actin, a wheat housekeeping gene (TraesCS1A01G020500). R, root; S, stem; L, leaf; FL, flag leaf; YS5, the 5 cm long young earfrom wheat plants at booting stage; YS10, the 10 cm long spike from wheat plants at heading stage; GR5, GR10, GR15, and GR20, the grains 5, 10, 15, 20, and 25 days post-anthesis, respectively. (B) Amplification products of the TaNAK1 transcripts obtained by RT-PCR; M, 250 bp DNA marker; 1, the products amplified with the gene-specific primer pair and the cDNA of wheat leaf as a template. (C) Subcellular localization of two protein isoforms TaNAK1.1 and TaNAK1.2 in wheat protoplasts. TaNAK1.1^N-terminal, the N-terminals containing both the kinase domain and the NB-ARC domain (1-353 aa) of TaNAK1.1; TaNAK1.2^N-terminal, the N-terminals containing both the kinase domain and the NB-ARC domain (1-421 aa) of TaNAK1.2; TaNAK1.1/1.2^C-terminal, the C-terminal containing NB-ARC domain and all subsequent sequences (521aa at the carboxyl end) of TaNAK1.1/TaNAK1.2; NLS-mCherry, the Nuclear Localization Sequence short peptide fused with mCherry (red fluorescent protein). The vector control (35S:GFP) and fusion protein vectors (35S: TaNAK1.1^N-terminal-GFP, 35S:TaNAK1.2^N-terminal-GFP, and TaNAK1.1/1.2^C-terminal-GFP) were each introduced into wheat protoplasts. GFP, the fusion proteins, and mCherry (red fluorescence) were observed with laser scanning confocal microscope; and Co-localization analysis of 35S:TaNAK1.2^N-terminal-GFP or TaNAK1.1/1.2^C-terminal-GFP (green) and NLS-mCherry (red) was observed by overlay. Bar = 10 μm. (D) The autophosphorylation activities of two protein isoforms TaNAK1.1 and TaNAK1.2 in vitro. Purified fusion proteins GST-TaNAK1.1^44-257 and GST-TaNAK1.2^44-306 were incubated independently with ATP and with or without CIAP in kinase reaction buffer, and their autophosphorylation activities were detected using Phos-tag Biotin (Wako, Japan). TaNAK1.1^44-257and TaNAK1.2^44-306 represents the kinase domains of TaNAK1.1 and TaNAK1.2, respectively. GST-TaNAK1.1^44-257 and GST-TaNAK1.2^44-306 indicate the protein kinase domains of TaNAK1.1 and TaNAK1.2 fused with GST, respectively. CIAP: Calf intestine alkaline phosphatase; Phos-tag Biotin: Biotinylated Phos-tag(Lumiprobe, USA); Anti-GST, GST antibody (Absin, Shanghai); : Phosphorylation.

Figure 3

The effects of two TaNAK1 splicing variants on the transition time from vegetative to reproductive growth of Arabidopsis. (A) The relative mRNA abundance of the TaNAK1 splicing variants in transgenic lines and wild-type Arabidopsis plants determined by real-time quantitative RT-PCR (qRT–PCR). Values are given as the mean ± SE; three independent biological replicates included. * and ** indicate significant differences at P < 0.05 and P < 0.01 levels, respectively, compared with the Col-0 using Student’s t test; ns represent no significant differences. (B) The phenotypes of bolting in different genotypes of Arabidopsis. (C) Bolting time in different genotypes of Arabidopsis. Bolting time (the time from sowing to the primary inflorescence reaching 1.0 cm in length) represents the transition from vegetative to reproductive growth in Arabidopsis. (D-G) The relative expression levels of flowering-related genes AtFLC, AtFT, AtLFY, and AtAP1 in different genotypes of Arabidopsis at 40 days post germination. Col-0: wild type Arabidopsis; EV: transgenic lines carrying empty vector p35S::Null; TaNAK1.1-OEs and TaNAK1.2-OEs: transgenic lines carrying expression vector p35S::TaNAK1.1 and p35S::TaNAK1.2, respectively. The data indicate means ± SE (n ≥ 12), and significance analysis was performed using Duncan one-way Anova; The different lowercase letters above the error bars indicate the different significance level at P < 0.05; ** and *** indicate significant differences at P < 0.01 and P < 0.001 levels, respectively, compared with the Col-0 using Student’s t test. ns, no significant difference.

Figure 4

The effects of two TaNAK1 splicing variants on plant architecture and seed yield related traits of Arabidopsis. (A) Images of different genotypes of Arabidopsis at maturity. Bar = 10 cm. (B, C) Plant height, the number of branches per plant of different genotypes of Arabidopsis. (D) Images of mature seeds from different genotypes of Arabidopsis. Bar = 0.5 mm. (E-J) Seed length, seed width, seed size, seed weight per plant, biomass per plant and harvest index of different genotypes of Arabidopsis. Col-0: wild type Arabidopsis; EV: transgenic lines carrying empty vector p35S::Null; TaNAK1.1-OE and TaNAK1.2-OE: transgenic lines carrying expression vector p35S::TaNAK1.1 and p35S::TaNAK1.2, respectively. The data indicate means ± SE (n ≥ 12), and significance analysis was performed using Duncan one-way Anova; the different lowercase letters above the error bars indicate the different significance at P < 0.05 level.