Phenotypic and transcriptomic characterization of a wheat tall mutant carrying an induced mutation in the C-terminal PFYRE motif of RHT-B1b

Abstract

Background: As central regulators of the gibberellic acid (GA) signaling pathway in plants, DELLA proteins function as growth repressors and affect diverse biological processes. The wheat RHT-B1b and RHT-D1b semi-dwarfing alleles, which encode GA-insensitive DELLA proteins, have been widely adopted in modern wheat varieties to improve lodging tolerance and harvest index. However, the molecular mechanisms by which DELLA modulates these responses in wheat remain largely unknown.

Results: We identified a tall tetraploid wheat mutant line carrying an induced missense mutation (E529K) in the PFYRE motif of RHT-B1b that partially suppressed the semi-dwarf phenotype. The height-increasing effect of RHT-B1b_E529K relative to RHT-B1b (19 cm or 21% increase) was significantly smaller than the effect of RHT-B1a (33 cm or 34% increase) relative to RHT-B1b in the same field experiment. The RHT-B1b_E529K mutation was also associated with length increases in coleoptiles, seedling shoots, and stem internodes relative to the RHT-B1b allele. We detected no significant differences in germination rate, seedling root length, tiller number, flag leaf size, spike length, or yield components. Using RNA-seq, we compared gene expression profiles of plants encoding RHT-B1b and RHT-B1b_E529K in coleoptile, first leaf, and elongating peduncles. We detected limited overlap among tissues of the genes differentially regulated by the two genotypes, and more genes upregulated (77%) than downregulated (23%) in RHT-B1b_E529K relative to RHT-B1b. These results suggest that the wheat DELLA protein affects the transcriptome in a tissue-specific manner and that the mutation mainly eliminates or reduces repression functions of the RHT-B1 protein. Our study identified distinct sets of potential DELLA direct or indirect target genes involved in cell wall and carbohydrate metabolisms, cell cycle/division, and hormone pathways.

Conclusions: We identified the hypomorphic RHT-B1b_E529K allele that confers an intermediate plant height and coleoptile elongation. This allele can be useful in rain-fed wheat breeding programs where the strong reduction in height and biomass associated with RHT-B1b has detrimental effects. Transcriptomic characterization of different tissues from the plants encoding RHT-B1b_E529K and RHT-B1b provided valuable information for identifying DELLA downstream GA response genes in wheat.

Keywords: DELLA; Plant height; RHT1; RNA-seq; Transcriptome; Wheat.

Fig. 1

Induced mutations identified in the distal region of the C-terminal domain of RHT-B1. Dark blue arrows indicate the locations of induced amino acid changes in RHT-B1b identified in this study (coordinates based on RHT-B1a, JF930278). The three mutations associated with increased height are highlighted in bold. The RHT-B1b_E529K mutation is highlighted in red. Light blue arrows indicate the locations of previously reported induced amino acid changes in RHT-B1c associated with increased height [34, 35]. Dark blue and light blue triangles indicate the positions of the Q64* premature stop codon in RHT-B1b and the 30 amino acid insertion in RHT-B1c, respectively. The PFYRE and SAW motifs [4] are underlined with solid and dashed lines, respectively

Fig. 2

Plant height of wheat plants homozygous for different RHT-B1 alleles. a Kronos*2/T4-934 plants homozygous for RHT-B1b and RHT-B1b_E529K. b Kronos*2/Gredho plants homozygous for RHT-B1b and RHT-B1a c Representative homozygous mutant lines and wild-type Kronos (see Fig. 1 for position and effect of the mutation in each line). d Average plant height of lines homozygous for RHT-B1b_E529K (T4-1405 and T4-934) and W605* (T4-3545). Different letters (a and b) indicate significant difference in a Tukey’s multiple comparison test at P < 0.05. Error bars indicate ± standard error of the means. a, b Field experiments, ^*** P < 0.0001 c, d Greenhouse experiments

Fig. 3

Effects of RHT-B1b_E529K on stem elongation. a Representative BC₁F₃ plants carrying homozygous RHT-B1b, heterozygous, and homozygous RHT-B1b_E529K alleles. b, c Internodes length of homozygous RHT-B1b (Wild), heterozygous (Het), and homozygous RHT-B1b_E529K plants (Mut) in F₂ and BC₁F₂ populations, respectively. Different letters (a, b, and c) indicate significant differences in a Tukey’s multiple comparison test at P < 0.05

Fig. 4

Effects of RHT-B1b_E529K on germination and seedling growth. a Germination rate. b Coleoptile, shoot and root length measured 2 weeks after sowing. NS: not significant. ***: P < 0.0001. Error bars indicate ±1 standard error. c Two-week-old seedlings of homozygous RHT-B1b and RHT-B1b_E529K BC₁F₃ sister lines

Fig. 5

Effects of RHT-B1b_E529K on GA sensitivity. An asterisk (*) indicates a significant (P < 0.05) difference in comparison with the control (GA₃ 0 μM) in a Dunnett’s test. The GA response index (GRI) was calculated by [100 × (length at GA₃ 10 μM)/(length at GA₃ 0 μM)]. Error bars indicate ±1 standard error

Fig. 6

Relationships among transcriptomes from four different tissues from homozygous RHT-B1b and RHT-B1b_E529K plants. a Hierarchical clustering of the 32 RNA-seq samples (two genotypes × four tissues × four biological replicates). b Principal component analysis of the 32 RNA-seq samples. RHT-B1b and RHT-B1b_E529K are indicated in blue and red, respectively. Tissues are indicated by shape (circle = coleoptile, square = first leaf, diamond = Z49 peduncle and triangle = Z52 peduncle)

Fig. 7

Differentially expressed genes (DEGs) and gene ontology (GO) enrichment analysis. a Number of DEGs between homozygous RHT-B1b (WT) and RHT-B1b_E529K (MT) plants in four tissues. b Significantly enriched GO terms among the DEGs between plants encoding RHT-B1b and RHT-B1b_E529K in Z49 peduncle. All GO terms are from the “Biological Process” category. Upregulated and downregulated GO terms are indicated in red and blue, respectively, with gradients indicating different levels of significance. The full list of 229 DEGs is provided in Additional file 1: Table S3