HvDep1 Is a Positive Regulator of Culm Elongation and Grain Size in Barley and Impacts Yield in an Environment-Dependent Manner

Abstract

Heterotrimeric G proteins are intracellular membrane-attached signal transducers involved in various cellular processes in both plants and animals. They consist of three subunits denoted as α, β and γ. The γ-subunits of the so-called AGG3 type, which comprise a transmembrane domain, are exclusively found in plants. In model species, these proteins have been shown to participate in the control of plant height, branching and seed size and could therefore impact the harvestable yield of various crop plants. Whether AGG3-type γ-subunits influence yield in temperate cereals like barley and wheat remains unknown. Using a transgenic complementation approach, we show here that the Scottish malting barley cultivar (cv.) Golden Promise carries a loss-of-function mutation in HvDep1, an AGG3-type subunit encoding gene that positively regulates culm elongation and seed size in barley. Somewhat intriguingly, agronomic field data collected over a 12-year period reveals that the HvDep1 loss-of-function mutation in cv. Golden Promise has the potential to confer either a significant increase or decrease in harvestable yield depending on the environment. Our results confirm the role of AGG3-type subunit-encoding genes in shaping plant architecture, but interestingly also indicate that the impact HvDep1 has on yield in barley is both genotypically and environmentally sensitive. This may explain why widespread exploitation of variation in AGG3-type subunit-encoding genes has not occurred in temperate cereals while in rice the DEP1 locus is widely exploited to improve harvestable yield.

Fig 1. Structure of the HvDep1 gene and the encoded polypeptide.

(A) Graphic representation of the barley whole genome shotgun contig bowman_contig_881907 comprising the HvDep1 coding sequence. 500-bp intervals are indicated with vertical bars. The coding sequence is shown as black boxes. The HvDep1 coding sequence consists of five exons and four introns. The ari-e mutant lines cv. Golden Promise and BW043 (ari-e.GP) show an insertion of a single nucleotide in exon 2 after bp 1508, and lines ari-e.39, ari-e.156 and ari-e.166 show a nucleotide exchange in exon 3 at bp 2007 after the ATG start codon (details shown in Table 3). (B) Putative functional domains in the 295 amino-acid HvDEP1 protein. Using TMPRED (ch.embnet.org/software/TMPRED_form.html) and PROSITE (expasy.ch/prosite) a transmembrane domain, a putative tumor necrosis factor receptor (TNFR) ⁄ nerve growth factor receptor (NGFR) family cysteine-rich signature and von Willebrand factor type C (VWFC) cysteine-rich modules were predicted for HvDEP1. In a previous study [7] the Arabidopsis AGG3 γ-subunit was predicted to contain similar domains.

Fig 2. Phenotype of barley breviaristatum-e (ari-e).

(A) Comparison of overall plant height between the wild type cv. Bowman (left) and ari-e.1 mutant NIL BW042 (right); scale bar: 10 cm (B) Spikes of barley lines Bowman, BW042 (ari-e.1), Maythorpe, Golden Promise (from left to right). Mutant lines are characterized by reduced awn length. (C) Seed shape of cvs. Maythorpe (left) and Golden Promise (right). (D) Gross morphology in the vegetative phase (from left to right: Bowman, BW042 (ari-e.1), Maythorpe, Golden Promise). Mutant lines appear more upright due to narrow leaf angles; scale bar: 10 cm.

Fig 3. Phenotype of ari-e mutant lines in a cv. Foma genetic background.

Barley lines Foma (A), ari-e.119 (B), ari-e.156 (C), ari-e.166 (D), ari-e.178 (E), ari-e.222 (F), ari-e.228 (G) during the vegetative growth phase when grown in the greenhouse. All pictures show three plants in one pot sown on the same day. Only ari-e.119, ari-e.156 and ari-e.166 show the characteristic erect phenotype of ari-e mutants and mutations in HvDep1 were only found in these lines. Scale bar: 10 cm

Fig 4. QTL profiles for 12 agronomic and yield related characters for chromosome 5H from the Derkado x B83-12/21/5 map.

QTL profiles for 12 agronomic and yield related characters for chromosome 5H from the Derkado x B83-12/21/5 map. The position of the Ari-e/Dep1 locus is indicated by the solid vertical arrow and the flanking SNP markers by vertical dashed arrows. BOPA1 SNPs 11_20265 and 11_20392 are placed at position 48.38 cM in the barley draft assembly [31]. The significance threshold (3.4) for a QTL in this population is indicated by the straight dashed horizontal line. SPW: Single Plant Weight of grain; TGW: Thousand Grain Weight; Spec_WT: specific grain weight [kg/hl]; MSW: Main Stem Weight, weight of grain from the tallest tiller of plants.

Fig 5. Grain yield measured in field trials in Denmark.

Values obtained for (A) grain number per plant and (B) grain weight per plant from field grown lines (summer 2014 in Denmark). The number of individuals considered for each line is given in Table 4. T-tests were performed to test for significant differences between respective wild type and mutant lines. Statistically significant differences are indicated by asterisks within the figure (threshold: * p < 0.05, ** p < 0.01, *** p < 0.001). Average values concerning grain number per plant and grain weight per plant respectively were 49.4% and 40.3% for BW042 and 51.7% and 39.7% for BW043 in comparison to Bowman and 81.6% and 66.7% for Golden Promise in comparison to Maythorpe.

Fig 6. Complementation of ari-e in Golden Promise with full length and truncated forms of HvDep1.

(A) and (B) Phenotype of Golden Promise, carrying the ari-e.GP loss-of-function allele, compared to (C) and (D) Golden Promise full-length pUBI:HvDep1 transformants and (E) and (F) Golden Promise pUBI: HvDep1Δ152 transformants. In the later, only the first 152 amino-acid residues of HvDEP1 are translated. Plants with the pUBI: HvDep1Δ152 transformation developed very few spikes and no fertile seeds.