BnERF114.A1, a Rapeseed Gene Encoding APETALA2/ETHYLENE RESPONSE FACTOR, Regulates Plant Architecture through Auxin Accumulation in the Apex in Arabidopsis

Abstract

Plant architecture is crucial for rapeseed breeding. Here, we demonstrate the involvement of BnERF114.A1, a transcription factor for ETHYLENE RESPONSE FACTOR (ERF), in the regulation of plant architecture in Brassica napus. BnERF114.A1 is a member of the ERF family group X-a, encoding a putative 252-amino acid (aa) protein, which harbours the AP2/ERF domain and the conserved CMX-1 motif. BnERF114.A1 is localised to the nucleus and presents transcriptional activity, with the functional region located at 142-252 aa of the C-terminus. GUS staining revealed high BnERF114.A1 expression in leaf primordia, shoot apical meristem, leaf marginal meristem, and reproductive organs. Ectopic BnERF114.A1 expression in Arabidopsis reduced plant height, increased branch and silique number per plant, and improved seed yield per plant. Furthermore, in Arabidopsis, BnERF114.A1 overexpression inhibited indole-3-acetic acid (IAA) efflux, thus promoting auxin accumulation in the apex and arresting apical dominance. Therefore, BnERF114.A1 probably plays an important role in auxin-dependent plant architecture regulation.

Keywords: BnERF114.A1; apical dominance; plant architecture; rapeseed (Brassica napus L.); seed yield; shoot branching.

Figure 1

The conserved structures of ERF114 proteins. (A) phylogenetic analysis and the structure diagrams of ERF114 proteins in Brassica napus and Arabidopsis. (B) alignment of the CMX-1 motif of ERF114 in B. napus, B. rapa, and B. oleracea. (C) alignment of the conserved AP2/ERF domain of ERF114 in B. napus, B. rapa, and B. oleracea. In (B,C), amino acids with black background indicate conserved amino acid residues across ERF114s.

Figure 2

BnERF114s expression profile. (A) relative expression levels of BnERF114s in rapeseed different tissues. Gene expression levels were normalised to the reference gene BnUBC21 (Gene ID: 106348550). Relative expression levels of BnERF114s were compared with the expression of BnERF114.C2 in flower. Each column represents the mean of three independent biological replicates, and the error bars indicate standard deviation. Different lowercase letter means significant difference at p = 0.05 level. (B) to (R), BnERF114.A1 expression patterns identified through histochemical GUS staining in Arabidopsis OE₁₁₄-46-3 transgenic line. (B) cotyledon (bar = 100 μm). (C) leaf primordium (bar = 20 μm). (D) root hair (bar = 50 μm). (E) root elongation zone (bar = 50 μm). (F) root tip (bar = 50 μm). (G–J) the youngest to the oldest rosette leaf, respectively (G–I, bars = 2 mm; J, bar = 9 mm). (K) new cauline leaves (bar = 1 mm). (L) mature cauline leaf (bar = 2 mm). (M) main inflorescence (bar = 2 mm). (N) branched inflorescence (bar = 1 mm). (O) flower (bar = 500 μm). (P) anther (bar = 200 μm). (Q) pot (bar = 2 mm). (R) wounded mature rosette leaf (bar = 2 mm).

Figure 3

Subcellular localisation of BnERF114.A1. Transient expression vector 35S::BnERF114.A1-eGFP was introduced into Nicotiana benthamiana leaf cells. The infected leaf was stained using DAPI as nuclear localisation marker. Bars = 30 μm.

Figure 4

Transcriptional activity and functional region of BnERF114.A1 tested by yeast system. GAL4 BD indicates that Modified P53 (a modified P53 protein only containing DNA binding domain), intact BnERF114.A1, and truncated BnERF114.A1 were separately fused with GAL4 BD. pGBKT7 is empty vector used as a control. Modified P53 is used as a negative control.

Figure 5

Effects of BnERF114.A1 ectopic expression on plant phenotype. WT, wild-type (Col-0) Arabidopsis; OE₃₅-18-1, transgenic line expressing BnERF114.A1-eGFP-GUS under the 35S promoter; OE₁₁₄-46-3, transgenic line expressing BnERF114.A1-GUS under the 114pro promoter. (A) phenotypes of OE₃₅-18-1 and OE₁₁₄-46-3 transgenic plants at 40 days old. (B) phenotypes of the main inflorescence and first rosette branches of OE₃₅-18-1 and OE₁₁₄-46-3 transgenic plants at 40 days old, in (A,B) Bars = 2 cm. (C) plant height of OE₃₅-18-1 and OE₁₁₄-46-3 transgenic lines at 30, 37, 44, and 51 days, each point represents mean ± SD of 5–10 independent individuals. (D) length of the main inflorescence in OE₃₅-18-1 and OE₁₁₄-46-3 transgenic lines at 37, 44, and 51 days; each column represents mean ± SD of 5–10 independent individuals. (E) number of first rosette branches in OE₃₅-18-1 and OE₁₁₄-46-3 transgenic lines at 37, 44, and 51 days. In (D,E), asterisks indicate significant differences at the p = 0.05 level, and “ns” indicates non-significant differences at the p = 0.05 level. (F) relative BnERF114.A1 expression in the main inflorescence of OE₃₅-18-1 and OE₁₁₄-46-3 plants at 40 days old; gene expression levels were normalised to the reference gene AtUBC21 (At5g25760); each column represents mean ± SD from three independent biological replicates, and each biological replicate included five individuals. Different lowercase letters indicate significant differences at the p = 0.05 level.

Figure 6

Yield-related traits of BnERF114.A1 transgenic plants. (A) number of siliques per plant. (B) number of seeds per silique. (C) thousand-seed weight (mg). (D) yield per plant (g). (E) biomass per plant (g). (F) harvest index. Values represent the data of 3 wild-type and 10 OE₃₅ transgenic plants. Asterisk indicates significant differences at the p = 0.05 level. “ns” indicates non-significant differences at the p = 0.05 level.

Figure 7

Relative expression levels of IAA-related genes and endogenous IAA level in main inflorescence of transgenic plants. (A) relative expression levels of four YUCCA genes (AtYUCCA1 [At4g32540], AtYUCCA2 [At4g13260], AtYUCCA4 [At5g11320], and AtYUCCA6 [At5g25620]); the expression level AtYUCCA1 in the wild-type (WT) being set as a unit. (B) relative expression levels of eight PIN family genes (AtPIN1 [At1g73590], AtPIN2 [At5g57090], AtPIN3 [At1g70940], AtPIN4 [At2g01420], AtPIN5 [At5g16530], AtPIN6 [At1g77110], AtPIN7 [At1g23080], and AtPIN8 [At5g15100]); the expression level of AtPIN1 in the WT being set a unit. (C) relative expression level of four AUX/LAX family genes (AtAUX1 [At2g38120], AtLAX1 [At5g01240], AtLAX2 [At2g21050], and AtLAX3 [At1g77690]), and the expression level of AtLAX2 in the WT being set as a unit. (D) relative expression levels of four PGP family genes (AtPGP1 [At2g36910], AtPGP2 [At4g25960], AtPGP4 [At2g47000], and AtPGP19 [At3g28860]); the expression level of AtPGP1 in the WT being set as a unit. Gene expression levels were normalised to the reference gene AtUBC21 (At5g25760). (E) endogenous IAA levels in the main inflorescences of the WT and transgenic plants at 40 days old. Each column represents mean ± SD from three independent biological replicates, and each biological replicate included five individuals. Asterisk indicates significant differences at the p = 0.05 level; “ns” indicates non-significant differences at the p = 0.05 level.