RING finger ubiquitin E3 ligase gene TaSDIR1-4A contributes to determination of grain size in common wheat

Abstract

Salt and drought-induced RING finger1 (SDIR1) is a RING-type E3 ubiquitin ligase that plays a key role in ABA-mediated responses to salinity and drought stress via the ubiquitination pathway in some plant species. However, its function in wheat (Triticum aestivum) is unknown. Here, we isolated a SDIR1 member in wheat, TaSDIR1-4A, and characterized its E3 ubiquitin ligase activity. DNA polymorphism assays showed the presence of two nucleotide variation sites in the promoter region of TaSDIR1-4A, leading to the detection of the haplotypes Hap-4A-1 and Hap-4A-2 in wheat populations. Association analysis showed that TaSDIR1-4A haplotypes were associated with 1000-grain weight (TGW) across a variety of different environments, including well-watered and heat-stress conditions. Genotypes with Hap-4A-2 had higher TGW than those with Hap-4A-1. Phenotypes in both gene-silenced wheat and transgenic Arabidopsis showed that TaSDIR1-4A was a negative regulator of grain size. Gene expression assays indicated that TaSDIR1-4A was most highly expressed in flag leaves, and expression was higher in Hap-4A-1 accessions than in Hap-4A-2 accessions. The difference might be attributable to the fact that TaERF3 (ethylene response factor) can act as a transcriptional repressor of TaSDIR1-4A in Hap-4A-2 but not in Hap-4A-1. Examination of modern wheat varieties shows that the favorable haplotype has been positively selected in breeding programs in China. The functional marker for TaSDIR1-4A developed in this study should be helpful for future wheat breeding.

Keywords: Triticum aestivum; Association analysis; RING E3 ubiquitin ligase; TGW; VIGS; functional marker; wheat.

Fig. 1.

E3 ubiquitin ligase activity of wheat TaSDIR1-4A. The fusion proteins MBP-TaSDIR1-4A and its mutant form MBP-TaSDIR1-4A^H244Y were assayed for E3 activity in the presence of E1 (from wheat), E2 (UBCh5b), and 6×His tag ubiquitin (Ub). The molecular masses of the marker proteins are shown (kDa). MBP was used as a negative control and MBP-AtSDIR1 was used as a positive control. Samples were resolved by 10% SDS-PAGE. An anti-Ub antibody was used to detect His tag ubiquitin (top image), and the anti-MBP antibody was used for detection of maltose fusion proteins (bottom image).

Fig. 2.

Nucleotide polymorphisms and development of a functional marker in wheat TaSDIR1-4A. (A) Schematic diagram of the TaSDIR1-4A structure. The ATG start codon was designated as position 1 bp. (B) Polymorphic sites were detected in the promoter regions of TaSDIR1-4A. (C) A dCAPS marker was developed based on the –395 bp single-nucleotide polymorphism (G/A). Digestion of the amplified 200-bp fragment with Age I produced fragments of 179 bp and 21 bp for accessions with the haplotype Hap-4A-1 (A), whereas this fragment was not digested in accessions with the haplotype Hap-4A-2 (G). Marker, 100-bp DNA ladder.

Fig. 3.

Comparisons of 1000-grain weight (TGW) in the two wheat TaSDIR1-4A haplotypes Hap-4A-1 and Hap-4A-2. (A) Comparisons in Population 2 (262 accessions) grown in 10 different environments: either at Changping (CP) or Shunyi (SY), and under drought-stressed (DS) or well-watered (WW) conditions, planted in 2010, 2011, or 2012. (B) Comparisons in Population 1 (doubled-haploid) grown in 10 different environments: either at CP or SY, and under DS, WW, or heat-stressed (HS) conditions, planted in 2015 or 2016. (C) Comparisons in Population 4 (348 modern cultivars) grown in three different environments: SY planted in 2010 or Luoyang (LY) planted in 2002 or 2005. Data are means (±SE), n=3. Significant differences between the haplotypes were determined using Student’s t-test: *P<0.05, **P<0.01, and ***P<0.001.

Fig. 4.

Phenotypic analysis of TaSDIR1-4A-silenced wheat and transgenic Arabidopsis. Virus-induced gene silencing (VIGS) of wheat was undertaken using a series of recombinant barley stripe mosaic virus (BSMV) vectors. (A) Relative expression of TaSDIR1-4A showing that transcription was lower in BSMV:TaSDIR1-4A plants than BSMV-free and BSMV:GFP plants. (B) Thousand-grain weight (TGW), (C) grain size, and (D) representative images of the grains. (E) Seed size in Arabidopsis plants in the Col-0 wild-type and T-DNA insertion mutant sdir1-1 backgrounds overexpressing (OE) TaSDIR1-4A. Vector, empty vector control. Significant differences between means were determined using one-way ANOVA followed by Tukey’s test (P<0.05).

Fig. 5.

Expression patterns of the wheat TaSDIR1-4A haplotypes Hap-4A-1 and Hap-4A-2. (A) Expression patterns of TaSDIR1-4A in different tissues of cv. H10 plants as measured by real-time PCR. S, spikes at flowering; FL, flag leaves; St, stems; N, nodes; RB, root bases; R30, roots from 0–30 cm depth; R50, roots from 30–50 cm; R70, roots from 50– cm; R90, roots from 70–90 cm; R100, roots from 90–100 cm. The values are relative to R90, which was set as 1. (B, C) Expression of Hap-4A-1 and Hap-4A-2 in (B) Population 1 and (C) Population 2. For each haplotype 12 accessions were randomly selected for measurement. (D) Expression patterns of TaSDIR1-4A following ABA treatment. Seedlings of cv. H10 at 2 weeks old were sprayed with 50 μM ABA solution and were sampled at 0 h (control, CK) and at 0.5–72 h. Expression is relative to the value in the control, which was set as 1. (E) Expression patterns of TaSDIR1-4A following NaCl treatment. Seedlings of cv. H10 at 2 weeks old were treated with 250 mM NaCl and were sampled at 0 h (CK) and at 0.5–72 h. Expression is relative to the value in the control, which was set as 1. In all cases GAPDH was used as the internal control. All data are means (±SE) of three biological replicates.

Fig. 6.

TaERF3 binds to the wheat TaSDIR1-4A Hap-4A-2 promoter region. (A) Schematic diagram of the GCC Box (–399 to –392 bp) in the Hap-4A-2 promoter. Letters in bold indicate the GCC Box regions, underlined letters indicate the GCC Box, letters in red are the single-nucleotide polymorphism sites (G/A), and the boxes indicate the 2-bp InDel sites. The fragments from the TaSDIR1-4A promoter regions of the cultivars H10 and L14 are shown, together with the 32-bp ACC Box from H10 (ACC-H10 Box), the same fragment but with A-to-G mutation (GCC-H10 Box), the 32-bp GCC Box from L14 (GCC-L14 Box), and the same fragment but with G-to-A mutation (ACC-L14 Box). (B) GAD-TaERF-BD activated expression in yeast of the LacZ reporter gene driven by the Hap-4A-2 (L14) promoter and the GCC Box (either from H10 or L14), rather than the Hap-4A-1 (H10) promoter and the 3×ACC Box (either from H10 or L14). (C) EMSA of the purified TaERF-BD protein and the GCC Box probe. Lane 1 contains biotin-labelled GCC-L14 Box probe, Lane 2 contains purified GST and biotin-labelled GCC-L14 Box probe, Lane 3 contains the TaERF-BD protein and biotin-labelled GCC-L14 Box probe, Lane 4 contains the TaERF-BD protein, biotin-labelled GCC-L14 Box probe, and 50× unlabeled (cold) GCC-L14 Box probe, Lane 5 contains the TaERF-BD protein, biotin-labelled GCC-L14 Box probe, and 200× cold GCC-L14 Box probe, Lane 6 contains the TaERF-BD protein, biotin-labelled GCC-L14 Box probe, and 500× cold GCC-L14 Box probe, Lane 7 contains the TaERF-BD protein, biotin-labelled ACC-L14 Box probe, Lane 8 contains the TaERF-BD protein, biotin-labelled GCC-L14 Box probe, and 500× unlabeled ACC-L14 Box probe, Lane 9 contains the TaERF-BD protein and biotin-labelled GCC-H10 Box probe, and Lane 10 contains the TaERF-BD protein and biotin-labelled ACC-H10 Box probe. The upper bands show that the TaERF-BD protein binds to the biotin-labelled GCC Box probe and the lower bands show the free probes. (D) Dual-luciferase assay of transformed tobacco leaves to examine the interaction between TaERFs and the TaSDIR1-4A promoter. Agrobacterium transformed with the vector pSoup harboring reporter and effector constructs, or with the empty vector (EV), were co-infiltrated into the leaves. Schematic diagrams of the effector and reporter constructs are shown. TaERF3 or TaERF115 was cloned into the effector construct pCAMBIA1300 and the promoter fragments from H10 or L14 were inserted into the reporter vector pGreen II 0800-LUC. Promoter activities are shown as the ratio of LUC to REN, and are relative to the control (CK) value, which was set to 1. Data are means (±SE) of three biological replicates.

Fig. 7.

Distribution of the TaSDIR1-4A haplotypes Hap-4A-1 and Hap-4A-2 in 10 wheat-producing regions across China. (A) Proportions of the two haplotypes in 157 Chinese landraces and (B) in 348 modern cultivars. Winter wheat regions: I, Northern; II, Yellow and Huai River valleys; III, Low and middle Yangtze River valley; IV, Southwestern; V, Southern. Spring wheat regions: VI, Northeastern; VII, Northern; VIII, Northwestern. Spring–winter wheat regions: IX, Qinghai–Tibet; X, Xinjiang.

Fig. 8.

Frequencies of the wheat TaSDIR1-4A haplotypes Hap-4A-1 and Hap-4A-2 in Population 4 (348 modern cultivars) together with their corresponding 1000-grain weights plotted against the decades in which the cultivars were released. The data for TGW are from Hao et al. (2011), n=3.