Pleiotropic function of SQUAMOSA PROMOTER-BINDING PROTEIN-BOX gene TaSPL14 on plant architecture of wheat

doi:10.21203/rs.3.rs-25293/v1

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Pleiotropic function of SQUAMOSA PROMOTER-BINDING PROTEIN-BOX gene TaSPL14 on plant architecture of wheat

https://doi.org/10.21203/rs.3.rs-25293/v1

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Background

SQUAMOSA PROMOTER-BINDING PROTEIN-BOX gene OsSPL14 from rice is evaluated as the major gene for ideal plant architecture consisting of few unproductive tillers, more grains per spike and high lodging resistance stems. However, the function of its orthologous gene TaSPL14 in wheat is still unknown.

Results

Here, we reported the similarity and variation between TaSPL14 and OsSPL14. Similar to OsSPL14, TaSPL14 knock-out mutants exhibited decreased plant height, spike length, spikelet number, thousand-grain weight. Different from OsSPL14, TaSPL14 had no effect on tiller number. Transcriptome analysis genes related to ethylene response were significantly decreased in young spikes of TaSPL14 knockout mutants, compared with wild type. TaSPL14 directly binds to the promoters of the ethylene response gene TaEIL1 (EIN3-LIKE 1), TaRAP2.11 (ETHYLENE-RESPNSIVE TRANSCRIPTION FACTOR 2.11) and TaERF1 (ETHYLENE-RESPNSIVE TRANSCRIPTION FACTOR 1) and activities their expression, suggesting that TaSPL14 might regulate wheat spike development through ethylene response pathway.

Conclusions

TaSPL14 had similar function with OsSPL14 in regulating plant height, spike length, spikelet number and thousand-grain weight of wheat, and had different function in tiller development. TaSPL14 might regulate spike development through TaEIL1, TaRAP2.11 and TaERF1, not TaDEP1.The elucidation of TaSPL14 will contribute to exploring the molecular mechanisms underlying plant architecture of wheat.

Plant Molecular Biology and Genetics

Plant Physiology and Morphology

Rice SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) gene OsSPL14 (Ideal Plant Architecture 1, IPA1) encodes a plant-specific transcription factor and is a new "Green Revolution" gene, which plays a critical role in regulating rice ideal plant architecture [1–3]. Therefore, the optimal increased expression of OsSPL14 in rice could confer ideal plant architecture including reduced tiller number, increased panicle primary branching, elevated lodging resistance of stems, at the same time increased thousand-grain weight [4–6]. Three natural alleles of OsSPL14 were identified from different high yield rice lines. The ipa1–1D allele contains a nucleotide substitution (C to A) affecting miR156 targeting and leads to the increased expression in shoot apex and the ideal plant architecture [7, 8]. WEALTHY FARMER’S PANICLE (WFP) epigenetic allele shows optimal increased expression of OsSPL14 in young spikes due to reduced DNA methylation in the promoter region, which has strong positive effect on primary branch and grain number [9]. The ipa1–2D epigenetic allele exhibits elevated expression of OsSPL14 due to tandem repeat sequences in promoter region producing an open chromatin structure, which controls tiller number, stem diameter and panicle primary branch number in a dosage-dependent manner [4]. In addition, base deletions in the exon of OsSPL14 by gene editing causes a frameshift mutation and the inactivation of OsSPL14 leads to dwarf phenotype with an extremely enhanced number of tillers and decreased plant height, panicle length and spikelet number [10]. Taken together, these findings show that OsSPL14 plays an important role in regulating ideal plant architecture in rice.

Previously, reported that OsSPL14 regulates ideal plant architecture through controlling downstream genes expression by directly binds the GTAC core motif in promoter [11]. One of target genes of OsSPL14, DENSE AND ERECT PANICLE1 (DEP1) controls rice panicle size [12, 13]. The expression level of DEP1 were significantly increased in shoot apices and slightly elevated in young panicles with higher expression of OsSPL14 [14]. The panicle length of RIL-IPA1/dep1 lines was significantly shorter than that in the RIL-IPA1/DEP1 lines, suggesting that OsSPL14 positively regulated DEP1 in adjusting panicle length in rice [12, 14]. OsSPL14 regulates tiller number by directly activating TEOSINTE BRANCHED1 (TB1), which is a negative regulator of tillering in rice [14, 15]. The expression level of OsTB1 were significantly raised in axillary buds in NIL-ipa1 line with higher expression of OsSPL14, and the RIL-ipa1/tb1 line could suppress the tiller phenotype of NIL-ipa1 plants, indicating that the direct activation of OsTB1 by OsSPL14 contributing to few tiller number of NIL-ipa1 plants [14, 16].

In wheat, only a couple of genes have been reported to affect plant architecture related traits: genes including tin1 (tiller inhibition gene 1), tin2 (tiller inhibition gene 2), tin3 (tiller inhibition gene 3) and ftin (fertile tiller inhibition) gene,are regulators for tiller inhibition [17–21]; Q gene is associated with spike compactness [22]. However, the effects of OsSPL14 ortholog, TaSPL14 on wheat plant architecture and yield is still largely unexplored. In this study, we aimed to analyze the function of TaSPL14 in developing ideal plant architecture of wheat. We constructed TaSPL14 knock-out mutants (taspl14) by using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system, and phenotypic analysis of mutants exhibited reduced plant height, spike length, spikelet number, thousand-grain weight but similar tiller number compared with wild type Fielder. Comparison of the transcriptome of young spikes between the taspl14 mutants and the wild type indicated that the genes involved in ethylene response were downregulated in mutants, and TaSPL14 could directly activate the expression of the TaEIL1, TaERF1 and TaRAP2.11 involved in ethylene signaling pathway. It is different from OsSPL14, TaSPL14 regulated spike length of wheat through regulating expression levels of genes associated with ethylene response, but not the TaDEP1. Therefore, our data provides new clues about exploring the functions and mechanisms of TaSPL14 in regulating plant architecture in wheat.

Identification of TaSPL14 in wheat

We annotated 56 sequences containing SBP domain from wheat genome sequence (TGACv1 version) [23]. Since common wheat (BBAADD) is a typical hexaploid species that generated from three diploid ancestral species Triticumurartu (AA), Aegilopsspeltoides (BB), and Aegilopstauschii (DD) [24–27]. The homoeologous genes from three subgenomes among these 56 sequences were grouped to 19 unique TaSPL genes, among which 10 had been previously isolated, named as previous report [28] (Table S1). A phylogenetic tree was performed based on the SBP-domain of 19 OsSPLs from rice and 19 TaSPLs from wheat [29–31]. According to the phylogenetic tree, the remaining 9 genes were named by orthologous genes of rice (Fig. 1A). We found three homoeologous genes including TraesCS5A02G265900, TraesCS5B02G265600 and TraesCS5D02G273900, designated TaSPL14-A, TaSPL14-B and TaSPL14-D, which showed high amino acid similarity with OsSPL14. Previous studies suggested that OsSPL14 was the target gene of OsmiR156 [7]. To verify whether miR156 could regulate TaSPL14 in vivo, we performed modified RNA ligase-mediated 5'-rapid amplification of cDNA ends (RLM-5'-RACE), and the results indicated that the TaSPL14 occurred cleavage at the 10th nucleotide from the 5' end of miR156 (Fig. 1B).

Three homoeologues genes of TaSPL14 were observed to be located on chromosome 5A, 5B and 5D, respectively, through amplifying from genomic DNA of Chinese Spring (CS) nullisomic-tetrasomic of chromosome group 5. The results showed the PCR amplification binds disappeared when the corresponding chromosome was deleted from CS lines (Fig. 1C). The expression levels of TaSPL14-A, TaSPL14-B, and TaSPL14-D among different tissues including seeding roots (SR), stems in jointing stage (SJ), seeding leaves (SL), grains at 4, 15, and 25 days after pollination (GDAP), young spikes with 5 mm (YS5), 10 mm (YS10), 20 mm (YS20) and 30 mm (YS30) in length were measured through reverse transcription quantitative real-time PCR (RT-qPCR) (Fig. 1D). The results indicated that the expression levels of TaSPL14-B or TaSPL14-D were higher than TaSPL14-A in measured tissues, TaSPL14-A and TaSPL14-D were mostly abundant in stems and young spikes, TaSPL14-B were mostly abundant in stems, leaves, seeds and young spikes, suggesting that three homoeologous genes of TaSPL14 might differently contribute to wheat development.

Functional comparison of TaSPL14 and OsSPL14

In order to investigate the biological function of TaSPL14 in wheat, we generated TaSPL14 knock-out mutant using the CRISPR/Cas9 system. In order to simultaneously knock out three homoeologous genes TaSPL14-A/B/D, we designed a specific sgRNA to target conserved region within first exon of the TaSPL14-A, TaSPL14-B and TaSPL14-D (Fig. 2A). The CRISPR/Cas9 vector pBUE411::sgRNA was transformed into wild type Fielder by Agrobacterium-mediated transformation [32]. Three independent transgenic T₀ plants were obtained. DNA sequencing for TaSPL14 revealed that three homoeologous genes mutated simultaneously with frameshift mutations in mutant line #5 and #12. The mutant line #13 showed a 3-base deletion of TaSPL14-5A without shifting mutation of protein coding region, while TaSPL14-5B and TaSPL14-5D were mutated simultaneously resulting code-shifting mutations (Fig. 2B). Homozygous T₂ generation plants #5 − 3, #12 − 4 and #13 − 4 were generated and planted in field.

We found all of three taspl14 mutant lines showed significantly decreased plant height and spike length compared to wide-type Fielder (Fig. 2C). Further observations revealed that the average length of rachis internode and spikelet number of taspl14 mutants were significantly decreased compared with Fielder (Fig. 2D). These results indicated that TaSPL14 affected wheat spike development through affecting both rachis internode elongation and spikelet formation. Thousand-grain weight (TGW) of mutant line #5 − 3, #12 − 4 and #13 − 4 was markedly decreased 17.3%, 22.88% and 23.7%g respectively compared to wild-type, indicating that the TaSPL14 influenced the grain weight of wheat (Fig. 2E). osspl14 (ipa1) mutants with a frame shift in protein and inactivation of OsSPL14, which caused rice mutants with notable decreased plant height, panicle length and number of flowers [10]. Moreover, thousand-grain weight increased in NIL OsSPL14^ipa1 with higher expression level of OsSPL14 [7]. Thus, our results indicated that TaSPL14 acts as a conserved regulator with OsSPL14 to pleiotropically regulate plant height, spike length, spikelet number and thousand grain weight.

However, different from OsSPL14, which is a regulator of rice tillering [5], TaSPL14 is not associated with tiller development in wheat. We found that tasp14 mutant line #5 − 3, #12 − 4, #13 − 4 showed similar tiller number as wild type Fielder in heading stage (Fig. 2F). These data indicated that TaSPL14 showed functional variation from OsSPL14 in tiller development.

Downstream genes affected by TaSPL14

To understand how TaSPL14 regulated spike development, RNA sequencing (RNA-seq) experiment was performed with young spikes (20–30 mm) of wild-type Fielder and mutant line taspl14-#13 − 4 with two biological replications. Totally 1103 and 228 genes were down- and up-regulated in taspl14-#13 − 4 compared with Fielder, respectively (Table S2). Further analysis of Gene Ontology of differentially expressed genes showed that upregulated genes were mainly enriched in the pathways related to red or far red light response and jasmonic acid response, while the downregulated genes were mainly enriched in the pathways related to meristem maintenance, meristems growth, cell proliferation and ethylene response (Fig. 3A, Table S3).

Among the down-regulated genes, we observed that 7 genes including TaERF1 (ETHYLENE-RESPNSIVE TRANSCRIPTION FACTOR 1), TaRAP2.11 (ETHYLENE-RESPNSIVE TRANSCRIPTION FACTOR 2), TaTCP (TEOSINTE BRANCHED1, CYCLOIDEA AND PCF TRANSCRIPTION FACTOR), TaRAP2-1A (ETHYLENE-RESPNSIVE TRANSCRIPTION FACTOR 1), TaEIN4 (ETHYLENE INSENSITIVE 4), TaRAP2-1D (ETHYLENE-RESPNSIVE TRANSCRIPTION FACTOR 1), TaEIL1 (EIN3-LIKE 1), which associated with ethylene response exhibited a statistically significant decreased expression in taspl14-#13 − 4 compared to Fielder (Fig. 3B). The down regulation of these genes in taspl14-#13 − 4 was further validated by performing RT-qPCR analysis (Fig. 3C-3I). Therefore, these results indicated that TaSPL14 regulated the expression of genes related to ethylene response.

It was reported that OsSPL14 was able to bind GTAC or TGGGCC/T core motif in promoter region of its target genes [14], which reminded us to detect whether TaSPL14 regulate genes related to ethylene response through binding to their promoters. The core GTAC motif was identified from promoter sequence of TaEIL1, TaRAP2.11 and TaERF1, we employed the electrophoretic mobility shift assays (EMSAs) to determine whether the TaSPL14 binds on these genes. Specific labeled and unlabeled probes were synthesized from promoter region of TaEIL1, TaRAP2.11 and TaERF1 respectively. As the results showed, the specificity bindings were observed in the combinations between TaSPL14- GST fusion proteins and labeled probes, and excessive molar of unlabeled probes prevented the band shift, but mutated unlabeled probes did not, indicating the TaSPL14 bound the GTAC core motif from the promoter of TaEIL1, TaRAP2.11and TaERF1 respectively (Fig. 4A, 4B, 4C). To confirm the regulatory effect of the TaSPL14 on TaEIL1, TaRAP2.11 and TaERF1 in vivo, the transcriptional activity assays by Agrobacterium tumefaciens-mediated transient infiltration were carried out in Nicotiana benthamiana leaves. We generated three plasmids containing the 2 kb promoter of TaEIL1 (pTaEIL1::LUC), TaRAP2.11 (pTaRAP2.11::LUC), TaERF1 (pTaERF1::LUC) driving the expression of the luciferase (LUC) reporter gene respectively (Fig. 4D, 4E and 4F). At the same time, we constructed a plasmid overexpressing TaSPL14 (35S::TaSPL14) as effector. These results confidently illustrated that co-expression of 35S::TaSPL14 with pTaEIL1::LUC, pTaRAP2.11::LUC and pTaERF1::LUC led to significantly elevates the LUC reporter activity compared with that in the empty vector controls. The above results suggested that TaSPL14 could directly and dramatically elevate expression of TaEIL1, TaRAP2.11 and TaERF1 (Fig. 4D, 4E and 4F).

OsDEP1, directly and positively regulated by OsSPL14, is an important regulatory gene that affects panicle architecture [12, 14, 33] In order to detect whether SPL14-DEP1 regulatory module is conserved between rice and wheat, we detected the expression of wheat TaDEP1 between taspl14-#13 − 4 and wild type Fielder. TaDEP1-A (TraesCS5A01G215100), TaDEP1-B (TraesCS5B01G208700) and TaDEP1-D (TraesCS5D01G216900) has been identified in wheat [34, 35]. Based on the results of RNA-seq, these three homoeologous genes were not differentially expressed in young spikes between mutant line taspl14-#13 − 4 and wild type Fielder, implying that TaSPL14 might regulate spike development through other genes such as related to ethylene response not TaDEP1 (Figure S1).

OsSPL14 is an essential component for regulating rice ideal plant architecture, including tiller development, panicle architecture and stems lodging resistant, substantially contributing to grain yield [14, 36]. However, the function and molecular mechanism of its orthologs, TaSPL14 is largely unknown, our study provided information about the role and mechanisms of TaSPL14 in regulating plant architecture of wheat.

Similar to OsSPL14, TaSPL14 acts as one of the major regulators for spike morphology. The taspl14 mutants showed reduction of spike length and spikelet number, but not altered the panicle branching (Fig. 2C, 2D). OsSPL14 regulates panicle architecture not only changing the panicle length and spikelet number, but also has strong effect on primary branching of rice [7, 9, 37]. Therefore, TaSPL14 and OsSPL14 showed the similar function in regulating length of spike and number of spikelet per panicle. It is worthy to note that OsSPL14 functions as a positive regulator of DEP1, which is an important component in regulating panicle architecture [14]. However, in our study the expression level of TaDEP1 was not altered in taspl14 mutant, compared with wild type (Figure S1), implying that TaSPL14 might regulate spike development not through DEP1, but rather other pathways. Here we revealed that the expression levels of 7 genes associated with ethylene response exhibited a significantly decrease in taspl14-#13 − 4 compared to Fielder and TaSPL14 could directly and dramatically elevate the driving activities of promoters of TaEIL1, TaERF1 and TaRAP2.11. This indicated that TaSPL14 might involve in spike development through regulating ethylene response pathway (Fig. 5).

Different from OsSPL14, TaSPL14 has no effect on tillering in wheat. The osspl14 mutants showed two contrasting phenotypes of tiller number in rice, osspl14 mutant plants with amino acid deletions in protein coding sequence and still maintained the activity of the protein, which showed fewer tillers than wild type [10]. Nevertheless, osspl14 mutants with a frame shift which may completely lost the action of OsSPL14, the mutants showed dwarf phenotype with an elevated number of tillers compare with wild type [10]. In this study, we found the tiller number of all taspl14 mutant lines was similar with wild type Fielder in the heading stage in the field (Fig. 2F), indicating the functional variation between OsSPL14 and TaSPL14 (Fig. 5).

OsSPL14 contains three yield-positive alleles IPA1 (ipa1–1D allele), WEALTHY FARMER’S PANICLE (WFP) epigenetic allele and ipa1–2D epigenetic allele, which all applied in rice breeding [4, 7, 9]. Introduction of the OsSPL14^ipa1 allele into Xiushui11 a japonica rice variety, increased the grain yield by ~ 10% in the test plot [7]. Introgression of OsSPL14^WFP allele into different rice varieties could significantly increase primary branches and grains per plant [9, 37–39]. For ipa1–2D epigenetic allele, JYZK-3 and JYZK-4 are two hybrid varieties bear ipa1-2D, markedly improved yield in comparison with the control variety at different locations [4]. Thus, it is meaningful to identify the natural variation related to optimal expression levels of TaSPL14 in young panicle, which could be used to design and generate high-yielding new varieties by using molecular assisted selection.

In this study, we indicated that TaSPL14 acts as a conserved regulator with OsSPL14 to pleiotropically regulate plant height, spike length, spikelet number and thousand-grain weight of wheat. And TaSPL14 showed functional variation from OsSPL14 in tiller development. TaSPL14 might regulate spike development through ethylene response genes, including TaEIL1, TaRAP2.11 and TaERF1, not TaDEP1. Our findings will be useful for programs that improve grain yield by manipulating wheat architecture.

SPL: SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE; IPA1:Ideal Plant Architecture 1; WFP:WEALTHY FARMER’S PANICLE; RIL:Recombinant inbred lines; TB1:TEOSINTE BRANCHED1; NIL:Near isogenic lines; tin:tiller inhibition gene; ftin:fertile tiller inhibition; CRISPR/Cas9:Clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9; SBP:SQUAMOSA BINDING PROTEIN; RLM-5'-RACE:RNA ligase-mediated 5'-rapid amplification of cDNA ends; PCR:Polymerase Chain Reaction; RNA-seq:RNA sequencing; RT-qPCR:Reverse transcription quantitative real-time PCR; EMSAs:electrophoretic mobility shift assays; GST:glutathione S-transferase; LUC:luciferase.

Availability of Data and Materials

The datasets generated and analysed during the current study are available in the NCBI (PRJNA629470) repository, https://www.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA629470.

Consent for Publication

Not applicable

Ethics Approval and Consent to Participate

Not applicable

Competing interests

The Authors declare that there is no conflict of interest.

Funding

National Natural Science Foundation of China (91935302) supported this work.

Authors’ contributions

Z.N, Q.S, Y.Y, and D.J designed the project and all experiments; J.C, K.L, W.S, and N.Z carried out the experiments and analysis experimental data; M.X, H.P, and Z.H responsible for the wheat transformation; J.C, Y.Y, and D.J contributed to article writing and reversion. All authors approved the contents of this manuscript.

Acknowledgments

We thank Dr. Qijun Chen (China Agricultural University) for providing the CRISPR-Cas9 vector (pBUE11), Dr. Rentao Song (China Agricultural University) for teaching transcriptional activity assays in N. benthamianathe and providing transient expression vector (pGreenII 0800-LUC).

Plant materials and growth conditions

Wild type Fielder and mutant taspl14 lines were grown in the experimental field of China Agricultural University in Beijing (39°57ʹN, 116°17ʹE) for seed reproduction, visible phenotypic and molecular analyses. Young spikes around 20–30 mm in length of wild type and mutants were collected, and immediately frozen in liquid nitrogen, then stored at -80 °C for following experiments. For molecular analyses, wheat materials were grown in artificial chamber. The conditions of artificial chamber is 45% humidity, 26/20℃ day/night temperatures, 3000lux illumination (Master GreenPower CG T 400W E40, PHILIPS).

Identification of TaSPL genes in wheat

To identify the novel SPL gene members in wheat, we downloaded SBP domain (PF03110) of SQUAMOSA-PROMOTER BINDING PROTEIN from Pfam (http://pfam.xfam.org/). All of sequences with SBP domain were obtained in wheat genome database (http://plants.ensembl.org/biomart/) based on Hiden Markov Model (HMM) profile with a cut of E-value < 1 × 10^− 5 [40–42]. Each predicted gene was confirmed by CDD website (http://www.ncbi.nih.gov/cdd) for conserved SBP domain analysis. Analysis of homoeologous genes was performed by using Ensembl Plant website (http://plants.ensembl.org/index.html).

Phylogenetic analysis of SPL genes

For phylogenetic analysis, 19 OsSPLs were obtained from GRAP [30]. Phylogenetic analysis was developed by using amino acid sequence of conversed SBP domain. We used CDD website to confirm the SBP domain sequence of SBP-box genes in rice and wheat, the ClustalW algorithm of MEGA5.02 to perform the multiple sequences alignment. A phylogenetic tree was constructed by using Neighbor-joining method, and bootstrapping with 1000 replicates. The accession number of OsSPLs and TaSPLs were shown in the Table S1.

Reverse transcription quantitative real-time PCR (RT-qPCR) analysis

According to the manufacturer, total RNA was isolated by using Trizol (Invitrogen, USA), and the first strand cDNA was synthesized by using the M-MLV Reverse Transcriptase kit (TaKaRa). We used the wheat ACTIN (TraesCS5B01G124100) gene as a reference for normalization. RT-qPCR were executed on the CFX96 real-time system (Bio-Rad) in 10 µl reactions containing 2 µl of each gene-specific primer, 1 µl of cDNA template, 2 µl of ddH₂O, and 5 µl of SYBR Premix EXTaq II (TaKaRa, Dalian, China). The RT-qPCR conditions were: 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 15 s, and 72 °C for 15 s, after that a melting curve was performed for each sample to analysis the specificity of the reactions. The relative expression levels were calculated using the 2^−ΔCT method normalized to CT value of wheat ACTIN gene, the RT-qPCR amplification of each sample was repeated three times. Gene-specific primers were shown in Table S4.

Vector construction and wheat transformation

We used CRISPR/Cas9 system to edit the genome sequence of TaSPL14 [43], and E-CRISP Design website (http://www.e-crisp.org/E-CRISP/designcrispr.html) to design small guide RNA (sgRNA). Later, two reverse complementary sgRNA sequences with BsaI site were synthesized. Intermediate vector (pCBC-MT1T2) connected to sgRNA sequences by using PCR, then, inserted into the terminal vector pBUE411 as previously described [43]. Plasmids were transformed into wild type Fielder by Agrobacterium-mediated transformation, EHA105 as Agrobacterium strain. The primers for vector construction were shown in Table S4.

RNA sequencing

We collected 5 young spikes with 20 mm-30 mm in length of wild type Fielder and taspl14 mutant line #13 − 4 for RNA sequencing. The extraction method of total RNA has been described before, and each RNA sample divided into two groups for two biological replicates. At least 5 µg total RNA of each replication was constructed cDNA libraries by using Illumina Poly-A Purification TruSeq library reagents, and performed on NovaSeq platform. The detailed analyze of RNA-seq data as previously described [44–46]. The differentially expressed genes between mutant line taspl14-#13 − 4 and the wild type Fielder were characterized by the Bioconductor package “edgeR” [47]. We used agriGO v.2.0 (cutoff of P < 0.05) to carried out GO analysis [48].

EMSA

The DNA-binding domain of TaSPL14 was expressed in BL21 Escherichia coli (Transgene) by cloning full-length of ORF sequence of TaSPL14 into the pGEX6P-1 with GST sequence. The detailed processes of induction and purification of proteins were as previously descripted [49, 50]. The labeled probes were 5'end with modified biotin, and the unlabeled probes without biotin modification in 5' end, which including competed probes that have same sequence as labeled probes, and mutant probes with changed core motif. All of probes were synthetized by company (Invitrogen), and to be double-stranded by cooling from 100 °C to room temperature in annealing buffer. We used the LightShift® Chemiluminescent EMSA Kit (Thermo) for EMSA, and the detailed processes of EMSA assays were as previously descripted [49]. Shifted complexes of labeled fragments and proteins, and free probes were visualized in nylon membrane by chemiluminescence camera (Tanon). Primers of EMSA were listed in Table S4.

Transcriptional Activity Assays in N . benthamiana

For reporter constructions, the 2 kb promoter sequences of TaEIL1, TaERF1 and TaRAP2.11 were cloned, and generated in the plant binary vector pGreen that has the luciferase reporter gene, respectively, and separately introduced into Agrobacterium tumefaciens strains GV3101 with pSoup. The full length of coding sequence of TaSPL14-B subgenome was cloned and generated into overexpression vector Supper1300 to generate effector construct, and introduced into Agrobacterium tumefaciens strains GV3101 [51]. The Agrobacterium tumefaciens strains GV3101 with reporters or effector were co-infiltrated N. benthamiana leaves. After 48 h infiltration, collected leaves and detected LUC activities by Dual-Luciferase Repoeter Assay System (Promega). Each experiment has three biological replications. The primers of transcriptional activity assays were shown in Table S4.

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Pleiotropic function of SQUAMOSA PROMOTER-BINDING PROTEIN-BOX gene TaSPL14 on plant architecture of wheat

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Abstract

Background

Results

Conclusions

Figures

Background

Results

Identification of TaSPL14 in wheat

Functional comparison of TaSPL14 and OsSPL14

Downstream genes affected by TaSPL14

Discussion

Conclusion

Abbreviations

Declarations

Availability of Data and Materials

Consent for Publication

Ethics Approval and Consent to Participate

Competing interests

Funding

Authors’ contributions

Acknowledgments

Methods

Plant materials and growth conditions

Identification of TaSPL genes in wheat

Phylogenetic analysis of SPL genes

Reverse transcription quantitative real-time PCR (RT-qPCR) analysis

Vector construction and wheat transformation

RNA sequencing

EMSA

Transcriptional Activity Assays in N . benthamiana

References

Supplementary Files

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