BnERF114.A1, a Gene Encoding an APETALA2/ETHYLENE RESPONSE FACTOR, Regulates Plant Architecture Through Blocking Auxin Efflux in Apex of Rapeseed Plant

doi:10.21203/rs.3.rs-476035/v1

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BnERF114.A1, a Gene Encoding an APETALA2/ETHYLENE RESPONSE FACTOR, Regulates Plant Architecture Through Blocking Auxin Efflux in Apex of Rapeseed Plant

https://doi.org/10.21203/rs.3.rs-476035/v1

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Plant architecture is very important for rapeseed breeding. Here, we reported an ETHYLENE RESPONSE FACTOR (ERF) transcription factor BnERF114.A1 of Brassica napus participating in plant architecture regulation. BnERF114.A1 is a member of ERF family group x-a, encoding a putative protein of 252 aa which consisting of an AP2/ERF domain and a conserved CMX-1 motif. BnERF114.A1 located in nucleus and had transcriptional activity with its functional region located in 142 aa ~ 252 aa of its C-terminus. The GUS staining analysis revealed that BnERF114.A1 highly expressed in leaf primordia, shoot apical meristem, leaf marginal meristem, and reproductive organs. Ectopic expression of BnERF114.A1 in Arabidopsis reduced plant height, increased branch numbers and silique numbers per plants, and finally increased seed yield per plant. Further investigation demonstrated that overexpression of BnERF114.A1 can inhibit IAA efflux and cause accumulation of auxin in apex, and arrest apical dominance in Arabidopsis. The findings suggested BnERF114.A1 could provide a candidate gene for rapeseed plant architecture molecular breeding.

Agronomy

Plant Molecular Biology and Genetics

Plant Physiology and Morphology

Rapeseed (Brassica napus L.)

BnERF114.A1

Plant architecture

Shoot branching

Apical dominance

Seed yield

we reported an ETHYLENE RESPONSE FACTOR (ERF) transcription factor BnERF114.A1 of Brassica napus L. participating in plant architecture regulation through blocking auxin efflux in apex of plant.

Since “Green revolution” occurred in wheat and rice which greatly increased crop yield since 1960s, much more attention has been paid to improve plant architecture to increase seed yield in rapeseed (canola, Brassica napus L.). Plant architecture is the three-dimensional organization of plant organs, adapting the plant for survival in different environments as well as affecting crop yield (Reinhardt and Kuhlemeier 2002). Crop breeders have focused on aerial architecture, including plant height; branch or tiller number and angle; leaf shape, size and angle; and inflorescence morphology (Wang and Li 2008; Wang et al. 2018; Li et al. 2019). Rapeseed is the most widely distributed and the largest sown edible oil crops in China, which provided more than 55% of edible oil in daily life. With the rapid development of national economy and the transfer of large number of rural labor force to the cities, agricultural farming methods are in urgent need of change, and simplification and mechanization has become the main direction of rapeseed breeding and industrial development in China (Luo et al. 2015). The ideal rapeseed plant architecture for mechanical harvesting is of dwarf, lodging resistance, tight plant (branch habit) suitable for dense planting, uniform ripening, cracking resistance and early maturing (Fu 2008; Fu and Zhou 2013). The branch habit of rapeseed plants includes branch position, branch angle and branch numbers, which are important in breeding of mechanized rapeseed varieties (Duan 2015). Therefore, investigation on branch characters and its genetic control mechanism of rapeseed is an important basis for genetic improvement of rapeseed suitable for mechanical harvesting.

In higher plants, the main stem shoot is derived from the primary shoot apical meristem (SAM), and the branches are differentiated from the meristem of axillary buds (AM) (McSteen and Leyser 2005). Branching occurs via a two-step process: initiation of an axillary meristem in each leaf axil to form a bud and subsequent bud outgrowth (Shimizu-Sato and Mori 2001). In some cases, the axillary bud is dormant as a result of complex interactions between endogenous developmental signals, including auxin, cytokinin and strigolactones, and environmental factors such as shade (Kebrom et al. 2010; Domagalska and Leyser 2011; Wang and Li 2011).

It has been demonstrated that plant architecture, a collection of genetically controlled agronomic traits, is mainly associated with the hormones and their signal transduction in plant. “Green revolution” occurred in rice and wheat mainly attributed to the reduced plant height that was caused by the deficiency of Gibberellin synthesis (sd1) and blocked degradation of DELLA proteins (Rht-B1b and Rht-D1b) (Peng et al. 1999; Sasaki et al. 2002). Indole-3-acetic acid (IAA) likewise, as the first discovered plant hormone, plays an important role in regulating plant architecture. It is well known that apical dominance mainly depends on IAA content in apex. IAA synthesis-deficient double mutants yuc1yuc4 and yuc2yuc6, triple mutants yuc1yuc2yuc6, yuc1yuc2yuc4 and yuc1yuc4yuc6, and quadruple mutant yuc1yuc2yuc4yuc6 displayed noticeable reduced plant height and loss of apical dominance (Cheng et al. 2006). Cytokinin (CTK) acts as an antagonistic hormone of apical dominance determined by IAA. IPT, cytokinin biosynthesis gene, promotes the outgrowth of axillary buds and inhibits auxin-dependent apical dominance (Shimizu-Sato et al. 2009).

APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) is one of the largest and the most important plant specific transcription factor superfamilies that encompasses 147 members in Arabidopsis (Nakano et al. 2006). All the members of this superfamily are characterized by harboring one or two essential 68-amino-acid repeat motif that was designated the AP2 domain and having DNA-binding activity (Jofuku et al. 1994; Ohme-Takagi and Shinshi 1995; Heyman et al. 2018). According to their structural features, AP2/ERF family members in Arabidopsis can be classified into four subfamilies: AP2 (18 members), RAV (six members), EREB-DREB (122 members), and AP2P-ISAMDD1 (a single member) (Nakano et al. 2006). The AP2 subfamily harboring two PA2 domains contains several key regulators that control different developmental processes during plant growth. RAV-type transcription factors contain a single AP2 domain and a second conserved DNA-binding domain designated B3 located at their C-terminus transcriptionally, and respond to touch-related stimuli (Kagaya et al. 1999; Kagaya and Hattori 2009). The largest subfamily EREB-DREB can be subdivided into 12 subgroups, namely I to X,VI-L and Xb-L, while the members of the X subgroup (ERF108 to ERF115) possess a hallmark of a conserved N-terminal sequence and participate in wound signaling and tissue repair (Nakano et al. 2006; Heyman et al. 2018).

It has been demonstrated that Arabidopsis AP2/ERF transcription factor gene EBE (At5g61890) belonging to the X subgroup and highly expressing in proliferating cells affects cell proliferation and axillary bud outgrowth as well as shoot branching (Mehrnia et al. 2013). A recent study revealed that ERF115, an X subgroup member of AP2/ERF transcript factors, functions in wound-induced stem cell division and sensitizes cell to auxin (Canher et al. 2020). In additions, ERF115 also functions as a repressor of adventitious root initiation by integrating the crosstalk of JA and CK (Lakehal et al. 2020). ERF109, another X subgroup member, has been confirmed that could be induced by wounding, and promoted auxin biosynthesis (Ye et al. 2020). Our previous investigation discovered that monosulfuron ester sodium (MES) could act as a new chemical hybridization agent for rapeseed, and low concentration of MES (10 ml per plant of 0.1 µg/ml) treatment could induce male sterility and increase branch numbers of rapeseed (Cheng et al. 2013; Li et al. 2015). Comparative transcriptome analysis between MES-treated and the control plants revealed that about 36% (542/1501) of differential expression transcripts associated with anther development of rapeseed were significantly up-regulated including some kinases and various transcript factors. One of such transcript factors is a integrase-type DNA-binding superfamily protein that is highly homologous with the EBE (At5g61890, also known as AtERF114) in Arabidopsis (Li et al. 2015), and therefore named as BnERF114. It was demonstrated that EBE (AtERF114) gene belongs to the X subgroup of ERF transcript factor family and contains AP2/ERF domain, which may affect cell proliferation, promote axillary bud generation and branch grow (Mehrnia et al. 2013). However, the characterization and biological function of BnERF114 in rapeseed (B. napus) remains unclear, and whether the phenotype of branch-increasing is related to the up-regulation of BnERF114 in rapeseed plants treated with low concentration of MES need to be further investigated.

To well understand the biological role of BnERF114 in growth and development of rapeseed, the homeologs of BnERF114 were isolated and their phylogenetic analysis was conducted in the present study. The molecular characterization and functional identification of BnERF114.A1 in growth and development of rapeseed were explored. Our results suggested BnERF114.A1 could regulate the property of plant shoot branching and probably serve as a candidate gene in rapeseed plant type breeding.

Plant materials and growth condition

B. napus cultivar Zhongshuang No.9 (ZS9) was planted in Yangling Regional Test Station of Crop Varieties, Shaanxi, China (N34.29◦, E108.06◦) at growing season 2017-2018. It was introduced from Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, China, and selfed for at least 10 generations prior to being used in the present investigation. Arabidopsis thaliana (Col-0) and transgenic plants were grown at 22℃under a 16-h light/8-h dark cycle (light intensity 6000 ∼ 9000 lux) and with the humidity of approximate 60% in phytotron.

Gene characterization and evolution analysis

The protein sequence of Arabidopsis AtERF114 (AT5G61890.1) was obtained from TAIR (http://www.arabidopsis.org/) website, and it was used as a query sequence to search for homologous genes in Brassica with PSI-Blast tool in NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). To confirm the genetic relationship of ERF114s from Brassica and Arabidopsis, evolution analysis was conducted by Mega 5 using conserved domain of AP2 (SMART accession number: SM00380). One hundred and twenty-two AP2/ERF family proteins from Arabidopsis were downloaded from TAIR web site and employed for constructing neighbor-joining (NJ) tree (Nakano et al. 2006).

Nucleic acid isolation

Total RNA of different rapeseed tissues and transgenic Arabidopsis was extracted using RNA extraction kit (E.Z.N.A. R Plant RNA Kit, OMEGA) according to the manufacturer’s protocol, and digested with RNA-free DNase I (Invitrogen) to remove DNA contamination. Genomic DNA was extracted from the young leaf samples of rapeseed or Arabidopsis plants according to the cetyltrimethylammonium bromide (CTAB) method (Porebski et al. 1997). The quality of the RNA and the DNA samples isolated were assessed by 2.0% and 0.8% agarose gel electrophoresis, respectively.

Cloning of the coding sequence and the promoter of BnERF114.A1 in rapeseed

For cDNA cloning, the first strand cDNA was synthesized using total RNA isolated from young pods of rapeseed ZS9 by GoScript™ Reverse Transcription System (Promega) according to the manufacturer’s protocol. The coding sequence (CDS) of BnERF114.A1 was isolated from the cDNA by polymerase chain reaction (PCR) using high-fidelity thermostable DNA polymerase KOD-FX-NEO (TOYOBO) and a primer pair BnERF114.A1-F and BnERF114.A1-R (Table S1). The PCR program was performed as pre-denaturation at 98℃ for 2 min followed by 40 cycles of 98℃ for 10 sec, 58℃ for 30 sec, 68℃ for 1 min, and final extension at 68℃ for 7 min. The PCR products were cloned into the pMD19-T vector (TakaRa, Dalian, China), and five clones randomly chosen were sequenced.

The promoter of BnERF114.A1 was cloned from genomic DNA of ZS9 using primer pair 114pro-F and 114pro-R (Table S1). The PCR program was performed as pre-denaturation at 98℃ for 2 min followed by 40 cycles at 98℃ for 10 sec, 60℃ for 30 sec, 68℃ for 1 min and final extension at 68℃ for 7 min. The products were cloned into the pMD19-T vector (TakaRa, Dalian, China), and five clones randomly selected were sequenced.

Subcellular localization of BnERF114.A1

To investigate the subcellular localization of BnERF114.A1, a primer pair 114SL-F and 114SL-R (Table S1) was used to clone the CDS region of BnERF114.A1, and a pGreen-35S::BnERF114.A1-eGFP expression vector was constructed using T4 DNA ligase (Takara) at 16℃ for 1h after EcoRI and SpeI double digestion. The constructed vector was confirmed by restriction analysis and sequencing. The recombinant vector was precipitated onto 1 μm gold particles (0.85 mg) and then bombarded into onion epidermal cells via a PDS21000/He particle gun (Bio-Rad) as described previously (Christou et al. 1988)，with pGreen-35S::eGFP as a control. Onion epidermis transient transformation was performed under 1100 psi rupture pressure with 9 cm target tissue distance. After cultured in darkness for 24h, onion epidermis was teared for observation by laser confocal microscope (Zeiss, Germany).

Transcription activity analysis of BnERF114.A1

To identify the transcriptional activity of BnERF114.A1 and its exact region, the full length cDNA sequence of BnERF114.A1, the 246 bp in the 3'-end of BnERF114.A1, the 177 bp encoding AP2 domain, and the 333 bp in the 5'-end of BnERF114.A1 were cloned by PCR, with primer pairs E756-F/E756-R, E246-F/E246-R, E177-F/E177-R, and E333-F/E333-R, respectively (Table S1). These target segments were separately introduced into pGBKT7 (Clontech) vector by T4 DNA ligase (Takara) at 16℃ for 1h after NdeI and EcoRI double digestion (Takara). The recombinant constructs were transformed into yeast strain AH109 using PEG-LiCl method (Gietz and Schiestl 2007), with empty pGBKT7 and the modified pGBKT7-p53 (only containing the DNA binding region) as negative controls, respectively. These colonies were screened on the SD/-Trp and SD/-Trp/-Ade/-His medium.

Construction of BnERF114.A1 overexpression vector and Arabidopsis transformation

CDS of BnERF114.A1 was introduced into a binary expression vector pCAMBIA3301 driven by CAMV 35S promoter (35S) by BamHI and SpeI double digestion and T4 DNA ligation (Takara), to generate 35S::BnERF114.A1-GUS vector. The expression cassette 114pro::BnERF114.A1-GUS was constructed by substituting the 35S promoter of 35S::BnERF114.A1-GUS vector for native promoter of BnERF114.A1 (114pro) through EcoRI and BamHI double digestion and T4 ligation (Takara). The resulted vectors 35S::BnERF114.A1-GUS and 114pro::BnERF114.A1-GUS were confirmed by sequencing, and transformed into A. thaliana (Col-0) by Agrobacterium tumefaciens-mediated floral-dip method (Zhang et al. 2006). Transgenic plants were screened by spraying 0.1% glufosinate (BASTA) on seedling leaves, and independent transgenic homozygous lines were obtained by self-breeding and spraying 0.1% glufosinate (BASTA) on seedling leaves. To assess the effects of BnERF114.A1 on transgenic plants, wild-type Arabidopsis and the obtained homozygous transgenic lines were grown at green houses as described above.

GUS staining analysis of BnERF114.A1 promoter activity

GUS activity was determined as described previously (Plesch et al. 2001). T₃homozygous transgenic plants of 114pro::BnERF114.A1-GUS were used for GUS activity analysis. Different tissues were incubated in GUS staining solution (50mM Phosphate buffer (pH=7.2), 0.01% Triton X-100, 2mM K₃Fe(CN)₆, 2mM K₄[Fe(CN)₆]·3H₂O, 10mM EDTA, 0.2mM X-Gluc) for 16 hours at 37°C. Wounded leaves were infiltrated in staining solution after injured with a knife and incubated at 37°C for 45 min. After treated with incremental concentration of ethyl alcohol, plant tissues were observed with stereomicroscope (Olympus SZ61) and camera (Canon 3000D).

Quantitative real-timereverse transcriptase-polymerase chain reaction

Quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was conducted to analyze the spatio-temporal expression patterns of BnERF114s in B. napus. The cDNA products of different tissues were normalized with housekeeping gene Ubiquitin-conjugating enzyme 21 of B. napus (BnUBC21，Gene ID: 106348550) as an internal reference gene. Three biological and three technical replicates were included for each RNA sample/primer combination for the qRT-PCR. Four primer pairs BnERF114.C2-qF/BnERF114.C2-qR, BnERF114.A1-qF/BnERF114.A1-qR, BnERF114.A6-qF/BnERF114.A6-qR, and BnERF114.C3-qF/BnERF114.C3-qR were used for analyzing expression levels of BnERF114.A1, BnERF114.C2,BnERF114.A6, and BnERF114.C3, respectively (Table S1).

The cDNA products of wild-type plants and transgenic lines were normalized using AtUBC21 (TAIR ID: At5g25760) as a reference gene to determine the expression level of auxin polar transport related genes and IAA synthesis genes. Three biological replicates were included. The qRT-PCR was performed in triplicate for each RNA sample/primer combination. The primer pairs used for qRT-PCR of AtPIN1~AtPIN8, AtAUX1, AtLAX1~AtLAX3, AtPGP1, AtPGP2, AtPGP4, AtPGP19, AtYUCCA1, AtYUCCA2, AtYUCCA4, and AtYUCCA6 were shown in Table S1. The program of qRT-PCR was as follows: pre-denaturation at 94℃ for 4 min, followed by 40 cycles at 94℃ for 20 sec, 60℃ for 20 sec, 72℃ for 20 sec and finally determined melting curve from 70℃ to 95℃ for 5 sec. The qRT-PCR was performed with GoTaq®qPCR master mix (A6001, Promega) on a QuantStudio™ 7 Flex Real-Time PCR System (Applied Biosystems). For each PCR, the specificity of the amplification was validated and the threshold cycle above background was calculated using Bio-Rad iCycler software, and PCR efficiency close to 100%.

The relative expression levels of the individual target genes were calculated by an improved double delta method (Pfaffl et al., 2002). Error bars of qRT-PCR data in all figures represented standard deviations, and significant differences were estimated at P=0.05 level using Student’s T test.

Endogenous IAA content analysis

The main inflorescences from 10 forty-day-old transgenic plants and wild type Arabidopsis were collected for IAA measurement, three biological replicates were included. IAA content was determined using Agilent 160 Infinity-6420 liquid chromatograph-mass spectrometer (Agilent Technologies) by Tsingtao Sci-tech innovation limited company.

Phenotype measurement and statistical analysis

To evaluate the effect of BnERF114.A1 on transgenic plant, plant height, the length of main inflorescence, numbers of the primary branches of transgenic plants and wild type plants were determined at 30, 37, 44, and 51 days after planting. Numbers of siliques per plant, number of seeds per silique, seed yield per plant and biomass per plant were evaluated at maturity. At least 10 plants of each independent transgenic lines or wild type were randomly selected for assessing these traits above mentioned. Statistical analysis was performed with unpaired t-test to evaluate significant difference.

Homology-based cloning and molecular characterization of BnERF114

To clone the orthologs of EBE (AtERF114) in B. napus, we blasted the full length amino acids of AtERF114 (AT5G61890.1) against Brassica Non-redundant protein sequences database of NCBI. As a result, eight highly homologous genes were obtained, with four genes from B. napus, two from B. rapa, and two from B. oleracea (Table S2). To identify the phylogenetic relationship of BnERF114s in B. napus, phylogenetic analysis and homologous analysis were conducted using AP2/ERF conserved domain of BnERF114s, BrERF114s, BoERF114s and Arabidopsis AP2/ERF superfamily genes. The result suggested that BnERF114.C2 derived from BoERF114.C2 of B. oleracea, BnERF114.A6 from BrERF114.A6 of B. rapa, BnERF114.C3 from BoERF114.C3 of B. oleracea. However, BnERF114.A1 may be derived from BrERF114.A2 (Figure S1, A). Phylogenetic analysis of BnERF114s and all ERF family members of Arabidopsis showed that BnERF114s were orthologs of AtERF114 (Figure S1, B) and belonged to the group X-a of AP2/ERF superfamily (Nakano et al. 2006). Consistent with AtERF114, all four BnERF114s contained two exons and one intron (Figure 1, A), and the encoding proteins contained an AP2/ERF domain (Figure 1, A & C) and a conserved motif CMX-1 (Figure 1, A & B). These results indicated that BnERF114s from B. napus were orthologs of AtERF114.

BnERF114.A1 expresses in proliferating tissue

To explore the spatio-temporal expression profile of BnERF114s, we measured their transcriptional levels in 13 different tissues of rapeseed cultivar ZS9. The results showed that the expression level of BnERF114.A1 was particularly higher than other three copies (BnERF114.C2, BnERF114.A6, and BnERF114.C3) in all tissues, indicating that BnERF114.A1 probably worked as a major gene among four BnERF114s. BnERF114.A1 highly expressed in root, cotyledon, flower, sepal, petal and pod, and had lower expression levels in other tissues such as rosette leaf, stem, small and middle bud, stamen and pistil (Figure 2, A). Considering the expression profiles of the BnERF114s during rapeseed development described above, BnERF114.A1was selected for subsequent functional studies.

To further detect the expression position of BnERF114.A1, we fused the -1836 bp ~ 0 bp of BnERF114.A1 promoter sequence (114pro) and the CDS of BnERF114.A1 with GUS. The recombinant construct 114pro::BnERF114.A1-GUS was transformed into Arabidopsis by Agrobacterium tumefaciens-mediated method. The GUS activity was analyzed in different tissues of transgenic Arabidopsis plants. The results exhibited that BnERF114.A1 remarkably expressed in leaf primordia, shoot apical meristem, leaf marginal meristem, tender cauline leaves (Figure 2, B~I), senescent leaves and cutting positions (Figure 2, J), and reproductive organs which including pistils and anthers (Figure 2, K~Q). In view of this, mechanical injury was performed on middle-aged leaves at 45 minutes before GUS staining. The result showed that mechanical injury could strongly and rapidly induce the expression of BnERF114.A1 (Figure 2, R). These results suggested that BnERF114.A1 may be involved in biological process of cell proliferation and damage signal response.

BnERF114.A1 mainly located in nucleus and had transcriptional activity

To confirm the subcellular localization of BnERF114.A1, the full length CDS of BnERF114.A1 from rapeseed cultivar ZS9 was isolated and fused with eGFP in frame to construct transient expression vector p35S::BnERF114.A1-eGFP. This transient expression vector was introduced into onion epidermal cells to express BnERF114.A1-eGFP fusion protein, with p35S::eGFP as a control. The result indicated that BnERF114.A1 mainly located in nucleus (Figure 3).

AP2 domain of BnERF114.A1 was predicted to be 83~141 amino acid. According to previous research (Nakano at al. 2006), this domain should be the DNA binding domain. To identify the transcription activity of BnERF114.A1 and active domain position, we introduced different fragments of the BnEFR114.A1 coding region, including full length CDS, N-terminus (246-), AP2 domain (-177-) and C-terminus of BnERF114.A1(-333) into pGBKT7 vector, respectively. The recombinant constructs were transformed into the yeast AH109 and screened on the yeast medium SD/-Trp and SD/-Trp/-Ade/-His. Yeast self-activation experiments proved that BnERF114.A1 had transcriptional activity and its active region was located in 142 aa ~ 252 aa of its C-terminus (Figure 4).

Ectopic expression of BnERF114.A1 in Arabidopsis reduced plant height and increased branch numbers

To explore the effects of BnERF114.A1 on plant architecture, we developed 35S::BnERF114.A1 (named OE₃₅ for short ) and 114pro::BnERF114.A1 (named OE₁₁₄ for short) transgenic Arabidopsis. At the beginning of bolting stage (about 30 days post planting), no differences of phenotype could be detected between wild type (Col-0) and transgenic Arabidopsis lines ectopically expressing BnERF114.A1. After 30 days post planting, it was observed that ectopic expression of BnERF114.A1 significantly inhibited the elongation of main inflorescence, which led to reduce plant height, with OE₃₅-18-1 as a representative of OE₃₅ lines and OE₁₁₄-46-3 as a representative of OE₁₁₄ lines shown in Figure 5 A~C because of their similar phenotypes among the same genotype. In additions, ectopic expression of BnERF114.A1 accelerated the emergence and outgrowth of lateral branches and first-order rosette branches of transgenic lines (Figure 5, D & E). OE₃₅ and OE₁₁₄ transgenic lines produced more rosette branches after 51 days of growing, compared with the wild type plants (Figure 5, E). The expression level of BnERF114.A1 in transgenic lines and wild type plants was determined, and the result showed that transgenic lines (OE35 and OE114) had significantly higher expression levels of BnERF114.A1 in the main inflorescences, rosette branch inflorescences and tender cauline leaves, compared to the wild type plants (Figure 5, F). These results suggested that BnERF114.A1 participated in regulating plant branching habit and inhibiting apical dominance of plants.

Ectopic expression ofBnERF114.A1 in Arabidopsis increased seed yield

To evaluate the effect of BnERF114.A1 on seed yield, we investigated yield-related traits of wild type Arabidopsis and transgenic lines overexpressing BnERF114.A1. Considering OE₃₅ lines with significant higher expression level of BnERF114.A1 than OE₁₁₄ lines, we compared the yield-related traits of OE₃₅ transgenic lines with those of the wild type plants. The OE₃₅ transgenic lines had a greater number of siliques per plant (270-320) than wild type Arabidopsis (170) (Figure 6, A), and had similar number of seeds per silique and thousand-seed weight as wild type plants (Figure 6, B & C). OE₃₅ transgenic lines showed much higher seed yield per plant (0.12-0.22g) and biomass per plant (0.8 -1.2g), compared with the wild type plant (0.06g and 0.5g, respectively) (Figure 6, D and E), this might be attributed to the ectopic expression of BnERF114.A1 greatly promoting the emergence and outgrowth of shoot branches (Figure 5, E), which increased number of siliques per plant (Figure 6, A).

Ectopic expression of BnERF114.A1 influenced the efflux of IAA in the main inflorescence of Arabidopsis

Apical dominance of plants is related to the synthesis and distribution of auxin in plants; Auxin synthesized from the apex of the plant is polar transported down to the lateral branches, thus inhibiting the growth of the lateral buds. It is reported that, decapitation of Arabidopsis induced the expression of AtERF114 in the five uppermost lateral branches beside the main inflorescences, consequently, the plants performed more lateral branches and cespitose phenotype (Mehrnia et al. 2013). In the present study, ectopic expression of BnERF114.A1 inhibited apical dominance of Arabidopsis, so we suspected that either auxin synthesis and/or auxin polar transport was blocked. To test our hypothesis, we determined the expression levels of IAA biosynthesis genes YUCCA1, YUCCA2, YUCCA4 and YUCCA6, which have been reported playing important roles in auxin-dependent apex dominance (Cheng et al. 2006). The results showed that expression of YUCCA1, YUCCA2, YUCCA4 and YUCCA6 was significantly decreased compared with wild type plants (Figure 7, A). We further determined the expression levels of IAA transport carriers (including PINs, AUX1/LAXs and PGPs) in the main inflorescences of transgenic lines and wild type Arabidopsis. The results showed that expression levels of PIN1, PIN3, PIN5, PIN6, AUX1/LAXs, PGP2 and PGP19 in transgenic lines significantly decreased in various degrees (Figure 7, B~D), however, the expression levels of PIN4 and PGP4 increased. These suggested that ectopic expression of BnERF114.A1 inhibited both auxin synthesis and the flux of IAA in Arabidopsis. So, we assumed that IAA was accomulated in the main inflorescences of transgenic lines. To further confirm this, endogenous IAA contents of main inflorescences of wild type and transgenic lines OE₃₅-18-1 was measured by liquid chromatogram. As we expected, the IAA contents of main inflorescences from transgenic lines dramatically increased, compared to that of wild type (Figure 7, E). These results indicated that ectopic expression of BnERF114.A1 influence endogenous IAA content of the main inflorescencesthe and flux of IAA in Arabidopsis.

Ideal plant architecture (ideotype) is an important objective in rapeseed breeding (Fu and Zhou 2013). Identification and characterization of more candidate genes for ideotype will contribute molecular designed breeding in this area. In this study, we characterized a new candidate ERF transcript factor gene BnERF114.A1 that belongs to a subgroup X-a member of ERF supfamily. Our results indicated that BnERF114.A1 located in nucleus and possed transcription activity. BnERF114.A1 expressed in proliferating tissues. Ectopic expression of BnERF114.A1 in Arabidopsis reduced plant height, inhibited apical dominance, enchanced shoot branch numbers, and finally, increased seed yield per plant and biomass. However, it did not affected harvest index. BnERF114.A1 regulated plant architeture by influence the efflux of IAA and endogenous IAA content of the main inflorescences of Arabidopsis.

Previous study showed that AtERF114 highly expressed in undifferentiated suspension culture cells and callus (Zimmermann et al. 2004). Menges et al. confirmed that AtERF114 had an elevated expression level at S phase of cell cycle, suggesting ERF114 related to cell proliferating and division (Menges et al, 2003). In Arabidopsis, AtEBE (AtERF114) prominently expressed in root tips, shoot apex, young leaves and reproductive organs (Mehrnia et al. 2013). In the present study, consistently, BnERF114.A1 generally expressed in active cell division tissues, such as leaf primordia, shoot apical meristem, leaf marginal meristem, tender cauline leaves and reproductive organs (Figure 4). In addition, previous studies showed that ERF114 and its homologous genes like ERF109/ERF115 could be induced by ROS signal which aroused by stress or wounding (Kong et al. 2018; Zhou et al. 2019; Canher et al. 2020). In the present study, strong GUS activity was also detected in senescent and wounded leaves (Figure 2, J and R), suggesting BnERF114.A1 also responsed to ROS signal.

AtEBE (AtERF114) stimulates axillary bud formation and outgrowth, while it's RNAi lines showed inhibited bud outgrowth and less branches than WT plant (Mehrnia et al. 2013). In the present study, overexpression of BnERF114.A1 in Arabidopsis remarkably inhibited apical dominance and promoted first-order lateral branches (Figure 5), indicating that BnERF114.A1 probably had similar function to AtEBE in promoting emergence and outgrowth of axillary buds. According to previous research, AtERF114 belongs to ERF transcription factor family X-a subgroup. This group is consist of eight members including ERF108~ERF115 (Nakano et al. 2006). Wound-induced JA could activate ERF109 that upregulate the expression of a tryptophan biosynthesis-related gene (ASA1) in auxin biosynthesis pathway, resulting de novo root regeneration (Zhang et al. 2019). In view of this, we determined the auxin level in BnERF114.A1 transgenic lines. The result showed that overexpression of BnERF114.A1 strikingly enhenced the accumulation of auxin in main inflorescens, suggesting that BnERF114.A1 had similar effect on auxin accumulation like AtERF109.

Auxin plays a vital role in regulating plant shoot branching and maintaining apical dominance (Leyser 2003). Long distance transport of endogenous IAA is performed in a polar manner depending on auxin polar transport carriers. It has been demonstrated that auxin efflux carrier PIN1 expressed in the vascular tissues and root primordial, regulating adventitious roots development and floral buds formation (Okada et al. 1991; Benková et al. 2003). In rice, down-regulated expression of OsPIN1 resulted in increased plant tiller number depending on auxin contents (Xu et al. 2005). Intercellular transport carrier PGP19 has the same location with PIN1 and can stablize PIN1 on cytomembrance, coordinately regulating auxin long-distance transport (Gälweiler et al. 1998; Blakeslee et al. 2007; Titapiwatanakun et al. 2009). In our study, ectopic expression of BnERF114.A1 caused decreased expression levels of PIN1, PIN3, PIN5, PIN6, AUX1/LAXs, PGP2 and PGP19 in Arabidopsis (Figure7, B~D), suggesting the efflux of IAA from apex to axillary bud being blocked. This was in line with the fact that IAA content was greatly increased in the main inflorescence of transgenic lines (Figure 7, E). The block of IAA efflux and accumulation of IAA in the main inflorescence resulted in the phenotype of reduced plant height and increased number of branches (Figure 5A).

YUCCAs play important roles in endogenous IAA biosynthesis. IAA synthesis-deficient double mutants yuc1yuc4, and yuc2yuc6, triple mutants yuc1yuc2yuc6, yuc1yuc2yuc4 and yuc1yuc4yuc6, and quadruple mutant yuc1yuc2yuc4yuc6 displayed noticeable reduced plant height and loss of apical dominance (Cheng et al. 2006). In the present study, ectopic expression of BnERF114.A1 resulted in decreased expression level of YUCCA1, YUCCA2, YUCCA4 and YUCCA6 (Figure 7, A), but increased IAA content in main inflorescence (Figure 7, E). We assumed that the increased IAA content might be the result of IAA accumulation when the efflux of IAA from apex to axillary bud being blocked. The decreased expression level of YUCCAs might be the result of feedback inhibition by IAA accumulation. This also explained that the blocked elongation of transgenic plant main stems occurred at approximately 10 days after bolting rather than the beginning of bolting stage.

In genernal, we isolated BnERF114.A1, an ortholog gene of AtERF114 in B. napus, which is expressed in leaf primordia, shoot apical meristem, leaf marginal meristem and reproductive organs. Ectopic expression of BnERF114.A1 reduced plant height, increased the number of shoot branching, and arrested apical dominance increased seed yield per plant in Arabidopsis. Ectopic expression of BnERF114.A1 regulates plant architecture through blocking auxin efflux in apex from apex to basal position. The findings suggested BnERF114.A1 was probably a valuable gene for rapeseed plant architecture molecular breeding.

Conflict of interest The authors declare that they have no conflict of interest

Acknowledgements

This work was financially supported by Open Project Fund of Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, P.R.China (Grant no. KF2018006) and National Natural Science Foundation of China (Grant no. 31801393).

Author contribution statement

Wang X, Hu S and Zhao H conceived and designed the experiments. Lyu J and Guo Y conducted the experiments and analyzed the data. Du C, Yu H and Liu L conducted parts of experiments. Guo L developed recombinant vectors. Lyu J wrote the draft of the manuscript, and Hu S and Zhao H revised the the manuscript. All anthors read and approved the final manuscript.

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BnERF114.A1, a Gene Encoding an APETALA2/ETHYLENE RESPONSE FACTOR, Regulates Plant Architecture Through Blocking Auxin Efflux in Apex of Rapeseed Plant

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