Exogenous strigolactones promote lateral root growth by reducing the endogenous auxin level in rapeseed

Journal of Integrative Agriculture 2020, 19(2): 465–482 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Exogenous strigolactones promote lateral root growth by reducing the endogenous auxin level in rapeseed MA Ni, WAN Lin, ZHAO Wei, LIU Hong-fang, LI Jun, ZHANG Chun-lei Oil Crops Research Institute, Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs/Key Laboratory of Crop Physiology and Production, Ministry of Agriculture and Rural Affairs, Wuhan 430062, P.R.China

Abstract Strigolactones (SLs) are newly discovered plant hormones which regulate the normal development of different plant organs, especially root architecture. Lateral root formation of rapeseed seedlings before winter has great effects on the plant growth and seed yield. Here, we treated the seedlings of Zhongshuang 11 (ZS11), an elite conventional rapeseed cultivar, with different concentrations of GR24 (a synthetic analogue of strigolactones), and found that a low concentration (0.18 µmol L–1) of GR24 could significantly increase the lateral root growth, shoot growth, and root/shoot ratio of seedlings. RNA-Seq analysis of lateral roots at 12 h, 1 d, 4 d, and 7 d after GR24 treatment showed that 2 301, 4 626, 1 595, and 783 genes were significantly differentially expressed, respectively. Function enrichment analysis revealed that the plant hormone transduction pathway, tryptophan metabolism, and the phenylpropanoid biosynthesis pathway were over-represented. Moreover, transcription factors, including AP2/ERF, AUX/IAA, NAC, MYB, and WRKY, were up-regulated at 1 d after GR24 treatment. Metabolomics profiling further demonstrated that the amounts of various metabolites, such as indole-3-acetic acid (IAA) and cis-zeatin were drastically altered. In particular, the concentrations of endogenous IAA significantly decreased by 52.4 and 75.8% at 12 h and 1 d after GR24 treatment, respectively. Our study indicated that low concentrations of exogenous SLs could promote the lateral root growth of rapeseed through interaction with other phytohormones, which provides useful clues for the effects of SLs on root architecture and crop productivity. Keywords: rapeseed (Brassica napus L.), strigolactones, lateral root growth, RNA-Seq, metabolic profiling analysis

1. Introduction

Received 8 October, 2018 Accepted 29 August, 2019 Correspondence MA Ni, E-mail: [email protected]; ZHANG Chunlei, Tel: +86-27-86739796, Fax: +86-27-86816451, E-mail: [email protected] © 2020 CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.1016/S2095-3119(19)62810-8

Oilseed rape (Brassica napus L.) is one of the major oil crops of the world and the second largest edible oil source for the human diet. Although the cultivated area of rapeseed and production of oilseed rape had reached 6.55 million ha and 13.33 million metric tons in 2018 in China (http://www. stats.gov.cn/tjsj/ndsj/), respectively, these levels were still insufficient to meet the increasing consumption demands (Lu et al. 2011). Therefore, the improvement of its seed yield is highly desirable. Seedling growth before the winter

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season is reported to be positively correlated with the seed yield (Diepenbrock 2000). Root architecture, defined as the spatial configuration of a root system, is an important factor that imparts the ability of a plant to acquire soil resources (Fang et al. 2013), and providing the water and nutrition for plant development and yield formation. Lateral root formation is critical for the development of root architecture. The lateral roots are formed from root pericycle cells adjacent to the protoxylem poles of the parent root and the lateral root primordium (LRP) is formed through several developmental stages with well-ordered cell divisions and cell differentiation (Strzalkal and Ziemienowicz 2011). The amount of lateral roots is influenced by diverse external and internal signals, the environmental, and most importantly, hormonal stimuli (López-Bucio et al. 2003; Malamy 2005; Ivanchenko et al. 2008). Phytohormones, particularly auxin and ethylene are involved in modulating root growth (Sun et al. 2010). Fukaki and Tasaka (2009) have suggested the role of indole-3-acetic acid (IAA) in the initiation of cell division in the pericycle, the promotion of cell division and the maintenance of cell viability in the developing lateral root. Up to now, it is well known that auxin signaling plays important roles in pericycle priming, initiation of cell division, and from the formation of LRP to lateral root emergence (Péret et al. 2013; Porco et al. 2016; Du and Scheres 2018). In other studies, it has been reported that enhanced ethylene synthesis, resulting from the application of low concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), promotes the initiation of lateral root primordia (Ivanchenko et al. 2008). However, Negi et al. (2008, 2010) have shown that the application of ethylene or the ethylene precursor, ACC reduces lateral root formation. Ethylene and auxin interact to regulate the initiation of lateral root primordia in the pericycle, as well as lateral root emergence and elongation (Ruzicka et al. 2007; Stepanova et al. 2007; Lewis et al. 2011). Actually, auxin can stimulate the production of ethylene, and ethylene modulates auxin biosynthesis and transport to affect root development (Swarup et al. 2007). All major processes of life depend on the differential expression of genes, which is largely controlled by the activities of transcription factors (Zhang et al. 2014). For instance, the AP2/ERF family, which includes the ethylene responsive transcription factor, is one of the largest transcription factor families in plants. AP2/ERF genes have been found to participate in various processes including plant growth and development, and their responses to various stresses (Jin et al. 2018). Trupiano et al. (2013) have reported that PtaERF003 function is linked to the auxin signal transduction pathway and is involved in lateral and adventitious root formation in Populus. Auxin (AUX)/IAA proteins act as transcriptional repressors that are required

to stimulate the lateral root primordia initiation at low doses of ACC (Ivanchenko et al. 2008; Lavenus et al. 2013). Strigolactones (SLs) are a new group of putatively carotenoid-derived terpenoid lactones isolated from root exudates, which can stimulate the seed germination of the root parasitic plant Striga (Yoneyama et al. 2008). SLs have a vital function in the regulation of shoot branching. GR24, a synthetic strigolactone, can either inhibit or stimulate shoot branching (Shinohara et al. 2013). Recently, significant progress has been made in understanding the role of SLs in shaping root architecture by auxin-SL cross talk, including the SL-mediated responses of primary root elongation, lateral root formation, adventitious root initiation, and secondary growth (Yoneyama et al. 2008; Brewer et al. 2013; Zwanenburg et al. 2016). GR24 acts as the positive regulator of primary root elongation in the strigolactone deficient and insensitive Arabidopsis mutants compared with wild type plants (Ruyter-Spira et al. 2011), while the lateral root growth was inhibited (Kapulnik et al. 2011). To date, the effects of SLs on the agronomically important rapeseed remain poorly understood. Zhongshuang 11 (ZS11) is an elite conventional rapeseed variety with many admirable traits, such as high yield and high oil content. It is widely cultivated in the Yangtze River valley region, China. It is noteworthy that the genetic background information of ZS11 has been extensively elucidated (Yang et al. 2014). In the present study, we found that low concentration of GR24 positively regulated ZS11 root tip initiation and lateral root length. These results emphasize the urgent need to further explore and understand the molecular mechanism of lateral root development in response to SLs. In this study, the differentially expressed genes (DEGs) assigned to pathways involved in the response to GR24 induction of lateral root growth were revealed. The results will provide important insight into the plants’ responses to hormonal stimuli and new data for future regulation of crop productivity.

2. Materials and methods 2.1. Plant materials, growth condition, and treatments The elite conventional winter rapeseed (B. napus L.) variety ZS11 was used in this experiment. Seeds originating from the inbred line ZS11 were surface-sterilized with 15% bleach for 10 min, and washed six times with sterile distilled water. Sterilized seeds were germinated on moist blotter paper in a growth cabinet at 22°C with a photoperiod of 16 h/8 h (light/dark). Three days after germination, approximately 1 000 seedlings were gently transferred to 1/2 Hoagland hydroponic fertilizer with oxygenation. Fifteen days later, 10 seedlings per replicate with three replicates were randomly

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sampled and separated into roots and shoots (ck_0  h). Then, other plants were treated with seven different concentrations of GR24 ranging from 0 to 18 µmol L–1, respectively. Each treatment was replicated three times with approximately 50 seedlings in each replicate. GR24 was initially dissolved in acetone (5.4 mg GR24/1 mL acetone) to give an 18 mmol L–1 stock solution, and this solution was further diluted with double-distilled sterile water to yield the indicated concentrations. All of the seedlings were grown at 22°C with a light regime of 16 h light and 8 h darkness. Light intensity was 250–350 μmol m–2 s–1.

Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Finally, the PCR products were purified (AMPure XP System) and library quality was assessed on the Agilent Bioanalyzer 2100 System (Zhang et al. 2014). The dataset-generated has been deposited in the National Center for Biotechnology Information (NCBI; project accession number: PRJNA 484313).

2.2. Investigation of plant growth

Reads were quality checked and trimmed for low quality regions and adaptor sequences with Trimmomatic (Bolger et al. 2014). Clean reads were mapped to the reference genome (Chalhoub et al. 2014) using Hisat2 (Kim et al. 2015). The expression level of each gene was measured by the fragments per kilobase exon model per million mapped reads (FPKM≥1) based on the number of uniquely mapped reads to eliminate the influence of different gene lengths and sequencing discrepancies on the gene expression calculation. Differential expression was performed with the DESeq2 (Love et al. 2014) R package using |log2(fold change)|≥1 and corrected P-value<0.05 as the threshold for significant differential expression. P-value was adjusted using the Benjamini and Hochberg’s approach to control the false discovery rate. The heatmap was plotted using the pheatmap R (ver. 3.3.0) package based on the mRNA expression. Identification and classification of transcription factors (TFs) in rapeseed were carried out by the iTAK Software (http://itak.feilab.net/cgi-bin/itak/index.cgi, ver. 1.6) (Zheng et al. 2016).

At 1, 4, and 7 d after the treatments, the shoot and root tissues of 10 plants were randomly sampled in each replicate. For the primary and lateral root formation, the primary root length of the seedlings with three replicates for each treatment was photographed and the images were analyzed with the computer image-analysis software WinRhizo Pro (Régent instruments). The separated roots and shoots were placed in the oven at 80°C to constant weight and measured. Data of root and shoot weight were analyzed using the statistical program SAS, and the analysis of variance (ANOVA) was followed by Fisher’s protected LSD test to identify homogenous groups within the means. Significant differences among treatments were considered at the P-value<0.05 level.

2.3. Root sample collection and RNA extraction Before SLs treatment, lateral roots were sampled with six biological replicates by mixing three seedlings per replicate (ck_0 h), and the samples were quickly frozen in liquid nitrogen. Mixed lateral roots grown in 1/2 Hoagland hydroponic fertilizer without GR24 solution (ck) and with 0.18 µmol L –1 GR24 solution (T) were sampled at 12 h (ck_12 h and T_12 h, respectively), 1 d (ck_1 d and T_1 d, respectively), 4 d (ck_4 d and T_4 d, respectively), and 7 d (ck_7 d and T_7 d, respectively) after treatment. The samples with three replications for each treatment were used for RNASeq. Total RNA was extracted using TRI reagent (MRC, Cincinnati, OH, USA) and digested with Turbo DNase enzyme (Ambion, Austin, TX, USA) according to the manufacturers’ instructions.

2.4. Library construction and sequencing A total amount of 3 μg RNA from each sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA

2.5. Mapping and DEGs analysis

2.6. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the DEGs GO and KEGG were performed on DEGs, which were compared to the whole genome background using the hypergeometric test with Benjamini and Hochberg false discovery rate correction at P-value<0.05.

2.7. Sample preparation and extraction for metabolites analysis To investigate the metabolite changes in the lateral roots after GR24 treatment, the samples with three replicates in each treatment were used for metabolites analysis. The freeze-dried roots were crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. Samples of 100 mg powder were weighed and extracted

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overnight at 4°C with 1.0 mL of 70% aqueous methanol. Following centrifugation at 10 000×g for 10 min, the extracts were absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250 mg, 3 mL; ANPEL, Shanghai, China, www.anpel.com. cn/cnw) and filtered (SCAA-104, 0.22 μm pore size; ANPEL, Shanghai, China, http://www.anpel.com.cn/) before LC-MS analysis.

2.8. HPLC-ESI-MS/MS analysis The sample extracts were analyzed using an LC-ESI-MS/ MS System (HPLC, Shim-pack UFLC SHIMADZU CBM30A system, www.shimadzu.com.cn/; MS, Applied Biosystems 4500 Q TRAP, www.appliedbiosystems.com.cn/). The analytical conditions by Chen W et al. (2013) were followed. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (Q TRAP) MS System. Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on an API 4500 Q TRAP LC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in a positive ion mode and controlled by Analyst 1.6 Software (AB Sciex Co., Canada). The ESI source operation parameters were shown by Wen et al. (2014). Metabolite structure was analyzed with the reference databases of MassBank (http://www.massbank.jp/), KNAPSAcK (http:// kanaya.naist.jp/KNApSAcK/), HMDB (http://www.hmdb.ca/), MoTo DB (http://www.ab.wur.nl/moto/), and METLIN (http:// metlin.scripps.edu/index.php). Metabolites were quantified relatively by calculating the area of each individual peak. Metabolites with dramatic differences in content were set with thresholds of variable importance, with VIP (variable importance in projection) value>1, fold change≥2 or fold change≤0.5.

2.9. Endogenous IAA analysis In this study, the plant materials were grown and treated (ck and T) as described above. The root samples were collected with three replicates at 12 h, 1 d, 4 d, and 7 d after treatment. The collected roots were immediately frozen with liquid nitrogen and stored at –80°C. The extraction and quantification of IAA were performed as described by Li et al. (2011). The calibration curve was linear in the concentrations’ ranges of 0.5–100 ng mL–1 and the R2 value was 0.9932. The solutions of IAA dissolved in methanol-0.05% formic acid (1:1, v/v) were separated by reversed-phase HPLC and scanned by tandem mass spectrometry (RP-HPLC/ESI-MS/ MS) (ABI 4000 Q-Trap, Applied Biosystems, Foster City, CA, USA) in the Multi Reaction Monitoring (MRM) mode with 50 ms dwell time. The mass spectrometry conditions and RP-HPLC setup and procedure were performed according to Li et al. (2011).

2.10. Quantitative real-time PCR validation of RNASeq data To validate the RNA-Seq data, a total of 12 candidate genes involved in the pathways of plant hormone signal transduction, nitrogen metabolism, MAPK signaling and peroxidase superfamily protein, and others, were chosen to design gene-specific primers (Appendies A and B). The primers were designed and PCR reactions were carried out according to Yang et al. (2004). Primer pairs were validated by standard curve analysis, and the expression levels of target genes calculated using the 2–ΔΔCT method, with actin as the internal control gene. Real-time PCR was carried out using a StepOnePlus™ Real-Time PCR System (Applied Biosystems) and Fast SYBR Green Master Mix (Applied Biosystems) to monitor double-stranded DNA synthesis in combination with 5-carboxy-X-rhodamine (ROX) as a passive reference dye. PCR reactions were carried out in duplicate using 7.5 pmol specific primers and approximately 5 ng cDNA in a total volume of 15 μL. The thermal profile for amplification was as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, 58°C for 30 s, and 60°C for 30 s. All reactions were performed with three independent biological replicates, and the expression levels calculated for each sample were based on three technical replicates. Data were analyzed using the ABI7500 Manager Software, and statistical analyses were conducted with SAS ver. 9.0 Software (SAS Institute, Cary, NC, USA) using Duncan’s multiple range test at the P<0.05 level of significance (Xu et al. 2015).

3. Results 3.1. GR24 treatment increased plant growth and biomass allocation The shoot dry weight, root dry weight, total dry weight, and root/shoot ratio increased extremely significantly (P˂0.01) among the different GR24 concentrations from 0 to 18 µmol L–1 at 1, 4, and 7 d (Fig. 1). The shoot dry weight, root dry weight, and total dry weight in ZS11 increased extremely significantly from 1 to 7 d at each GR24 concentration from 0 to 1.8 µmol L–1. For example, F=1 893.950, P˂0.01 for shoot dry weight, F=334.471, P˂0.01 for root dry weight and F=3 406.906, P˂0.01 for total dry weight at the concentration of 0.18 µmol L–1, respectively. Remarkably, the significant increase of the root/shoot ratio from 1 to 7 d indicates that GR24 treatment has a strong effect on root growth (F=5.341, P˂0.05). In contrast, the dry weight decreased significantly when the concentration reached 18 µmol L–1 (F=94.015, P˂0.01 for shoot dry weight, F=76.762, P˂0.01 for root

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Fig. 1 Effects of GR24 on the dry weight of the seedlings at 1, 4, and 7 d after GR24 treatment (n=10) in Zhongshuang 11 (ZS11). A, shoot dry weight. B, root dry weight. C, total dry weight. D, root/shoot ratio. All experiments were repeated three times and the data represent mean±SE.

dry weight, and F=99.540, P˂0.01 for total dry weight, respectively). In addition to the above-mentioned results on dry matter biomass, our analysis showed that GR24 had no significant effect on the primary root length, whereas it had a significant effect on the number of root tips and lateral root length. The root tip numbers significantly increased (F=6.646, P˂0.01) after treatment with 1.8×10–4 to 1.8 µmol L–1 GR24 compared with the control at the 1, 4, and 7 d (Fig. 2-A). Meanwhile, the change trend of lateral root length demonstrated that GR24 affected the root development significantly from 1 to 7 d after either treatment (Fig. 2-B). For instance, F=67.582, P˂0.01 for root tip numbers and F=107.739, P˂0.01 for lateral root length at the concentration of 0.18 µmol L–1. However, at 18 µmol L–1, GR24 significantly suppressed the lateral root elongation (F=62.671, P˂0.01 for root tip numbers and F=16.236, P˂0.01 for lateral root length, respectively). The results of root morphology showed that low concentrations of GR24 acted as a positive regulator for lateral root development.

3.2. Significantly DEGs were found at 12 h and 1 d after treatment In the present study, root/shoot ratio increased significantly

under GR24 treatment, and we hypothesized that the effect on the root growth was more pronounced. Therefore, the transcriptome analysis was conducted to better understand transcriptional changes in response to GR24 treatment and how these changes varied over time. In this study, gene expression accounting for the lateral root growth with the 0.18 µmol L–1 GR24 treatment was analyzed. Lateral root samples from ZS11 at five time points (0 h, 12 h, 1 d, 4 d, and 7 d) after GR24 treatment and the control conditions were used to construct nine libraries for sequencing. About 50 million raw reads were obtained for each library, and over 98% of the sequences remained after the data filter process. Approximately 65% of the clean data could be mapped to the rapeseed reference genome (Appendix C). The Pearson correlation analysis showed that the three biological replicates had good correlation (Appendix D). To identify candidate genes that respond to GR24 treatment in rapeseed roots, we focused on the DEGs identified by comparing the gene expression levels between treatment and control groups at each time point. Finally, 1 886, 3 237, 1 180, and 661 genes were up-regulated, while 415, 1 389, 415, and 122 genes were down-regulated in GR24 treatment samples compared to the control at 12 h, 1 d, 4 d, and 7 d, respectively. Clearly, the expression levels of most of the genes increased when plants had the GR24

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treatment (Fig. 3). The Venn diagram demonstrates the numbers of DEGs and unique up- and down-regulated genes at each time point (Fig. 4). Most of the DEGs were uniquely associated with a specific time point (Fig. 4-A). For example, there were 3 237 up-regulated genes at 1 d, and more than 90% of DEGs were found to be increased only at 1 d (Fig. 4-B).

In addition, only six common DEGs at four time points and only one gene were down-regulated (Fig. 4-A and C). Because there were many more DEGs between GR24 treatment and control at 12 h and 1 d than at 4 and 7 d, 300 genes that were expressed differentially at 12 h and 1 d were used to further analyze their functions.

3.3. Three expressed patterns of DEGs 1d

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During the early stages after GR24 application, 300 DEGs exhibited diverse expression patterns at 12 h and 1 d. As the hierarchical clustering of the expression levels showed (Fig. 5), the DEGs were divided into three different subclusters. In the top subcluster, 87 genes were

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Fig. 3 Differentially expressed genes (DEGs). The T_12 h vs. ck_12 h indicates a comparison between the gene expression in the T_12 h library with that in the ck_12 h library. T_12 h, T_1 d, T_4 d, T_7 d, ck_12 h, ck_1 d, ck_4 d, and ck_7 d indicate lateral root tissues after GR24 treatment (T) and the control (ck) at 12 h, 1 d, 4 d, and 7 d, respectively. DEGs were confirmed based on whether the |log2(fold change)|≥1 and P-value<0.05.

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Fig. 2 Effects of GR24 on the lateral root length (A) and number of root tips (B) of seedlings at 1, 4, and 7 d after GR24 treatment (n=10) in Zhongshuang 11 (ZS11). All experiments were repeated three times and the data represent mean±SE.

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Fig. 4 Overview of analysis of differentially expressed genes (DEGs) identified by pairwise comparisons of the eight root transcriptomes, T_12 h, T_1 d, T_4 d, T_7 d, ck_12 h, ck_1 d, ck_4 d, and ck_7 d, which indicate lateral root tissues after GR24 treatment (T) and the control (ck) at 12 h, 1 d, 4 d, and 7 d, respectively. A, Venn diagram of DEGs between GR24 T and ck at the four time points. B, Venn diagram of up-regulated genes at the four time points. C, Venn diagram of down-regulated genes at the four time points. The individual and overlapping areas in Venn diagrams represent the numbers of specifically expressed and co-expressed genes between the different time points.

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up-regulated at 12 h and then down-regulated at 1 d. In the middle subcluster, 94 genes were up-regulated at the two time points. In the bottom subcluster, most genes were down-regulated at both 12 h and 1 d (Fig. 5-A). The expression trend profiles of the DEGs were illustrated by a cluster analysis based on the K-means methods (Fig. 5-B). The expression trend of DEGs in subcluster 1 demonstrated the opposite pattern to that in subcluster 2, and the 87 genes in subcluster 3 were expressed at their highest levels at 12 h in the GR24 treated samples.

3.4. GO and KEGG analysis indicated the molecular and cellular events involved in lateral root development We used GO assignments to classify the functions of the 300 DEGs in pairwise comparisons of the cDNA libraries between GR24 treatments and control, and between 12 h and 1 d. Three GO categories were presented in Fig. 6. In the biological process (BP) category, the most abundant GO terms were “metabolic process” (GO: 0008152) with

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98 genes and “response to stress” (GO: 0006950) with 36 genes. In the cellular component (CC) category, “cell periphery” (GO: 0071944) consisted of 29 genes and was the most abundant, followed by “extracellular region” (GO: 0005576) with 21 genes. Similarly, in the molecular function (MF) category, “catalytic activity” (GO: 0003824) was the most abundant category with 132 genes, followed by “oxidoreductase activity” (GO: 0016491) with 37 genes. We detected 11 cell wall related genes that were significantly differentially expressed in the root tissue at 12 h and 1 d. These DEGs, such as WRKY33, pectinesterase, chitinase, and xyloglucan endotransglucosylase/hydrolase, are involved in FAD-binding and some structural constituents of the cell wall. A total of 2 301, 4 626, 1 595, and 783 DEGs between GR24 treatment and the control at 12 h, 1 d, 4 d, and 7 d (P-value˂0.05) were categorized into 64, 49, 44, and 43 functional groups respectively, that were clustered in the BP, CC, and MF categories (Appendix E). Metabolic process and response to stimulus were dominant terms in the category of BP. Membrane (GO: 0016020) and cell

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Fig. 5 Hierarchical cluster analyses of 300 common differentially expressed genes (DEGs) between GR24 treatments (T) and the control (ck) at 12 h and 1 d. A, this map shows the genes of |log2(fold change)| values for the GR24 effects on the root. The ck_12 h group indicates an average gene expression level of three replicates in the ck_12 h library, and the same notation describes as T_12 h group, ck_1 d group, and T_1 d group. B, the clustering of the DEGs. The three major clusters obtained by the K-means algorithm, represent down-regulated (1), up-regulated (2), and transient (3). FPKM, fragments per kilobase exon model per million mapped reads.

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Cellular component

Molecular function

Fig. 6 Gene Ontology (GO) analyses of the 300 common differentially expressed genes (DEGs) after GR24 treatment at 12 h and 1 d. The DEGs were assigned to three groups, including biological process, cellular components, and molecular function.

periphery were dominant terms in the main category of CC. Catalytic activity and oxidoreductase activity were dominant in the main category of MF, and were similar to those at 12 h and 1 d. The DEGs associated with different functional categories clearly indicated the molecular and cellular events that were involved in rapeseed lateral root development. To identify the biological pathways which responded to GR24 treatment, DEGs were mapped to the reference canonical pathways in the KEGG (Appendix F). The DEGs between GR24 treatment and the control at the 12 h and 1 d were assigned to 51 KEGG pathways. The greatest representation by unique genes was the metabolic pathways (ko 01100) with 49 members and biosynthesis of secondary metabolites (ko 01110) with 31 members. It was interesting that the MAPK signaling pathway (ko 04016) with nine members, phenylpropanoid biosynthesis pathway (ko 00940) with 10 members, nitrogen metabolism (ko 00910) with three members and plant hormone signal transduction (ko 04075) with eight members were also mapped (Fig. 7). For example, PRX52, a peroxidase superfamily protein, was involved in metabolic pathways including the biosynthesis of secondary metabolites pathway and the phenylpropanoid biosynthesis pathway. WRKY33 was assigned to the MAPK signaling pathway, and it was involved in responses to various abiotic stresses, especially salt stress. AXR3, the AUX/IAA transcriptional regulator family protein, was involved in plant hormone signal transduction (Appendix F). Additionally, we investigated the up- and down-regulation of the special DEGs assigned to KEGG pathways (Fig. 8).

The results powerfully demonstrated that many more upregulated genes were assigned to these pathways at 12 h and 1 d. These annotations provide a valuable resource for investigating specific processes, functions, and pathways involved in GR24 induced lateral root formation of rapeseed.

3.5. The TFs were mostly up-regulated In the current study, many transcription factors were differentially expressed between the GR24 treated and untreated rapeseed at the four time points. These TFs include AP2/ERF, AUX/IAA, WRKY, and several others. The numbers of some significantly differentially expressed TFs were shown in Table 1. The results indicated that the number of total differentially expressed TFs increased from 12 h to 1 d, and decreased at the 4 and 7 d. The five significantly differentially expressed TF families also demonstrated high counts at 12 h and 1 d (Fig. 9). Moreover, the overwhelming majority genes in TFs were up-regulated. For instance, AP2/ERF, the ethylene responsive TF, included a total of 36 members with 32 up-regulated and four down-regulated at 12 h. Some primary auxin response genes were clearly up-regulated by GR24 treatment. We detected AUX/IAA transcription factors at the 1 d time point, and 11 of them were up-regulated, including IAA3, IAA11, IAA17, IAA19, IAA26, IAA27, and IAA28, while another four members exhibited lower expression level. The WRKY transcription factors modulated the root system in a positive way, and 37 of them were all up-regulated at 12 h. Interestingly, many of the genes showed different temporal and spatial

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Pathway enrichment Zeatin biosynthesis Plant-pathogen interaction Plant hormone signal transduction Phenylpropanoid biosynthesis –log10Q-value 5

Pentose and glucuronate interconversions

Pathway name

Nitrogen metabolism

4 3

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MAPK signaling pathway - plant

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Glycolysis/Gluconeogenesis

Gene number 10

Glutathione metabolism

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Fatty acid elongation

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Cysteine and methionine metabolism Biosynthesis of secondary metabolites Arginine biosynthesis Amino sugar and nucleotide sugar metabolism Alanine, aspartate and glutamate metabolism 0.01

0.02

0.03

Rich factor

Fig. 7 The pathway enrichment of the 300 common differentially expressed genes (DEGs) after GR24 treatment at 12 h and 1 d. The size of circle represents the number of genes. The heatmap represents the value of –log10Q-value.

expression patterns (Fig. 10). For example, 20 genes in the top cluster for AP2/ERF transcription factor were expressed at significantly higher levels at 12 h and then expressed at lower levels at the subsequent time points. The results confirmed that the genes involving the AP2/ERF, AUX/IAA, NAC, MYB, and WRKY transcription factors were mostly up-regulated at 12 h and 1 d after GR24 treatment.

3.6. Metabolic profiling analysis proved that the metabolic pathway, phenylpropanoid biosynthesis pathway, and tryptophan metabolism pathway played important roles in the regulation of lateral root growth The extreme diversity of metabolites found in plants makes them the ideal models for dissecting the biosynthetic pathways of the metabolites and the regulation of these metabolic pathways. Further evidence for the role of GR24 in the lateral root growth was investigated by metabolic profiling analysis. The number of differential metabolites

was the highest at 1 d (Fig. 11-A), which was consistent with the trend of change in the numbers of DEG at the four time points by RNA profiling analysis. Twenty differential metabolites up-regulated at 1 d, such as L-Isoleucine and L-Pyroglutamic acid, and 25 metabolites were downregulated, such as IAA and cis-zeatin (Appendix G). The concentrations of endogenous IAA in the lateral root tissues at different time points and treatments were shown in Fig. 11-B. The endogenous IAA concentrations decreased significantly by 52.4 and 75.8% at the 12 h and 1 d, when comparing the GR24 treatment with the control, respectively. Nevertheless, the concentrations had no significant differences at 4 and 7 d. The differential metabolites were assigned to 29 KEGG pathways at the 1 d, and the greatest representation was metabolic pathways (ko 01100) with seven members, followed by biosynthesis of secondary metabolites (ko 01110) with five members. The conjoint analysis of transcript and metabolic profiling was conducted and a Pearson correlation

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Fig. 8 Up-regulated and down-regulated differentially expressed genes (DEGs) assigned to the Kyoto encyclopedia of genes and genomes (KEGG) pathways of metabolic pathways (A), biosynthesis of secondary metabolites (B), plant hormone signal transduction (C), nitrogen metabolism (D), mitogen-activated protein kinases (MAPK) signaling pathways (E), and tryptophan metabolism (F) between lateral root tissues after GR24 treatment (T) and control (ck) at 12 h, 1 d, 4 d, and 7 d. Table 1 The number of differentially expressed transcription factors at 12 h, 1 d, 4 d, and 7 d Family AP2/ERF AUX/IAA bHLH bZIP C2H2 C2C2 GARP HB HSF LOB MADS MYB NAC Others TAZ WRKY

Up 32 1 0 3 18 3 1 1 14 0 1 14 32 2 0 37

12 h Down 4 4 3 2 2 4 0 1 0 0 1 7 1 7 1 0

Total 36 5 3 5 20 7 1 2 14 0 2 21 33 9 1 37

Up 17 11 22 3 15 17 9 36 2 15 9 42 20 8 2 14

1d Down 13 4 10 6 12 15 3 6 2 1 9 36 8 5 1 10

Total 30 15 32 9 27 32 12 42 3 16 18 78 28 13 3 24

Up 17 3 16 7 7 2 0 9 3 9 1 16 12 21 4 2

4d Down 0 1 3 5 4 1 3 4 0 0 0 2 0 1 0 3

Total 17 4 19 12 11 3 3 13 3 9 1 18 12 22 4 5

Up 4 0 4 0 6 0 3 1 2 3 2 6 7 7 4 1

7d Down 1 0 0 0 0 1 0 2 0 0 0 0 0 0 0 0

Total 5 0 4 0 6 1 3 3 2 3 2 6 7 7 4 1

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coefficient (PCC)>0.8 was adopted. The results proved that the metabolic pathways, phenylpropanoid biosynthesis pathway and tryptophan metabolism pathway played important roles in the regulation of lateral root growth after GR24 treatment (data not shown).

of lateral root were very sensitive to GR24 treatment. Moreover, the functional categories “membrane”, “cell periphery”, and “cell wall” were over-represented, which was consistent with the reports by Zhang et al. (2014). As noted, although SLs were applied to the roots, it could move from the roots into the shoots (Khosla and Nelson 2016). SLs are coordinators of shoot and root development and mediators of plant responses to environmental conditions (Koltai 2011b). In our experiments, when the roots were treated with GR24, not only did the root weight increase, but the shoot biomass also increased. Moreover, we found that the oxidative stress under salinity in rapeseed was alleviated by GR24 treatment (Ma et al. 2017). These results indicated that the rapid growth of the seedling will hopefully lead to obtaining high productivity as reported by Diepenbrock (2000). In the recent years, SLs were discovered to be the communication signals in the plant, soil, and rhizosphere, and they have different activities in plant development and in the rhizosphere (Kapulnik and Koltai 2014; Koltai 2014). All of the prior studies suggest the application of SLs can be very useful in agriculture.

3.7. Verification of the gene expression through RT-qPCR

4.2. The lateral root development mediated by auxin and ethylene signaling

As shown in Fig. 12, based on the verification results of RTqPCR, the expression trends of 10 out of 12 genes (83%) were consistent with those of RNA-Seq results, which means the RNA-Seq data were credible.

In higher plants, lateral root formation is critical for the development of root architecture. SLs have been found to repress lateral root initiation in Arabidopsis (Ruyter-Spira et al. 2011; Kapulnik et al. 2011; Foo and Reid 2013), which is inconsistent with our results. Although both Arabidopsis and rapeseed belong to the Brassicaceae family, there are a great number of genetic, developmental, and physiological differences between these two plant species (Yang et al. 2014). We speculate that the low concentrations of GR24 in our experiment could be regarded as high amounts for Arabidopsis treatment, which were found to inhibit lateral root development. In the current study, GR24 treatments with low concentrations (1.8×10–4 to 1.8 µmol L–1) promoted the lateral root development while 18 µmol L –1 GR24 showed an inhibitory effect. The regulation pattern found here is similar to the reports that different concentrations of GR24 treatments can either inhibit or stimulate shoot branching, depending on the auxin transport status and the concentration in the treated plant (Shinohara et al. 2013). An explanation for 10 nmol L–1 GR24 promotion of shoot branching is that it systemically removes PIN1 from plasma membranes, and auxin transport capacity is also systemically reduced (Shinohara et al. 2013). Lateral root formation is initiated when pericycle cells accumulate auxin, thereby acquiring founder cell (FC) status and triggering asymmetric cell divisions, giving rise to a new primordium.

AP2/ERF

AUX/IAA

MYB

NAC

WRKY

Count

150 100 50 0 0.5

1

4 Time (d)

7

Fig. 9 Differential expression of transcription factors (TFs) of rapeseed change over time under GR24 treatment. The five significantly changed TF families are shown.

4. Discussion 4.1. Plant growth in response to GR24 treatment in rapeseed The plant root is an important organ for absorbing water and nutrients from the soil. Root growth and differentiation in plants have been inextricably linked with plant hormones (Saini et al. 2013). In this study, the root dry weight of rapeseed increased significantly when treated with GR24 concentrations ranging from 1.8×10–4 to 1.8 µmol L–1, in comparison with the control condition. Because there were no significant differences in the primary root length, the increased dry weight was attributed to the positive effects on the lateral root growth. The remarkable increase of root tip numbers and root length confirmed these results. Root growth depends on two developmental processes: cell division and elongation in the root meristem region (Beemster and Baskin 1998). The significant differences of lateral root tips and root lengths appeared at 1 d after treatment and showed that the cell division and elongation

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Group ck T

Group Time BnaA03g06460D BnaAnng34430D BnaA05g24040D BnaCnng66390D BnaA05g17950D BnaC08g31830D BnaC02g24050D BnaC03g23960D BnaA01g08370D BnaA03g49310D BnaC07g41360D BnaA09g24190D BnaC05g24890D BnaA07g19100D BnaC05g28390D BnaA05g01110D BnaC04g51650D BnaA05g34370D BnaC02g04540D BnaC09g43890D BnaAnng33660D BnaA05g37410D BnaC05g21870D BnaA09g51620D BnaCnng01510D BnaC04g11030D BnaA08g00130D BnaA03g32550D BnaC03g73730D BnaA05g26240D BnaC05g40360D BnaA03g02640D BnaA05g24050D BnaA06g22900D BnaC03g50570D BnaA05g18430D BnaC03g03740D BnaA10g20110D BnaC08g34030D BnaA03g49460D BnaA04g14380D BnaC03g08880D BnaC04g36420D BnaC09g09210D BnaC02g00990D BnaCnng09850D BnaA03g19960D BnaC04g52880D BnaAnng11180D BnaC03g40490D BnaCnng55590D BnaA01g26760D BnaCnng63190D BnaA07g28000D BnaA07g24270D BnaC06g43410D BnaA01g28930D BnaC03g39160D BnaA10g22680D BnaC09g47250D BnaA07g10300D BnaA02g33910D BnaC02g42720D BnaA09g41400D BnaAnng05950D BnaC07g34980D BnaC01g36040D BnaC07g13550D

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h_group h_group d_group d_group d_group d_group d_group d_group

BnaA03g20950D BnaC04g03870D BnaA05g15470D BnaA05g20050D BnaA08g16220D BnaC03g60650D BnaA09g16520D BnaA03g31510D BnaA08g08310D BnaAnng07060D BnaCnng05080D BnaA05g27930D BnaC05g42130D BnaA07g29400D BnaA03g04290D BnaC07g08350D BnaA02g34370D BnaA04g19700D BnaAnng13220D BnaC02g43290D BnaC04g44080D BnaC02g05060D BnaC03g05820D BnaA07g06750D BnaA10g27750D BnaCnng78540D BnaA04g27050D BnaC04g51210D BnaA06g01090D BnaC03g49530D BnaA09g04470D BnaC09g50850D BnaC04g50030D BnaA10g04090D BnaC05g04210D BnaC09g20090D BnaCnng60520D BnaCnng05070D BnaAnng21280D BnaC01g10100D BnaA06g40170D BnaC09g45000D BnaC09g49920D BnaCnng67070D BnaA06g35500D BnaC08g18660D BnaA02g25110D BnaA08g08300D BnaC07g16190D BnaA06g33420D BnaA07g12050D BnaA07g35130D BnaA06g10760D BnaA02g06490D BnaC03g26480D BnaA04g13590D BnaC04g35800D BnaA09g39350D BnaA10g12950D BnaCnng71740D BnaA07g12040D BnaA07g30130D BnaC06g33570D BnaA03g34290D BnaC07g46770D BnaA01g23940D BnaA03g22100D BnaA07g13990D BnaA08g15790D BnaC03g61270D BnaA03g19580D BnaA09g05710D BnaCnng47540D BnaC09g05370D BnaA01g27570D BnaC01g35070D BnaA05g23130D BnaC09g04110D BnaA06g27540D BnaA09g04650D BnaC03g48820D BnaC07g43950D

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Group Time BnaA02g31030D BnaA08g02410D BnaC04g00310D BnaC04g49810D BnaC07g47230D BnaA01g05500D Novel04300 BnaA03g48160D BnaC04g05170D BnaC04g01540D BnaA05g11870D BnaA06g40630D BnaC03g24960D BnaAnng39820D BnaC03g60600D BnaAnng01980D BnaC02g01720D BnaA03g04160D BnaC04g35770D BnaA04g13570D BnaC03g05760D BnaA07g24310D BnaA09g20470D BnaA05g01410D BnaAnng32610D BnaA04g22040D BnaA03g17820D BnaCnng66020D BnaC02g22950D BnaC03g21360D BnaC03g67380D Novel01067 BnaA09g13370D BnaC09g13680D BnaA07g16850D BnaC06g15910D BnaC07g49530D BnaC09g44020D BnaA05g34850D BnaC04g06800D BnaA04g12540D BnaA04g02560D BnaC09g36400D BnaA09g07010D BnaC01g13500D BnaC09g11790D BnaA09g11350D BnaCnng11190D BnaA04g23480D BnaC08g27340D BnaA09g35840D BnaA09g55920D BnaA06g23750D BnaC03g49470D BnaC07g43490D BnaCnng13810D BnaC04g19430D BnaC04g47350D

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BnaA06g38670D BnaAnng26800D BnaC09g03530D BnaA02g01290D BnaA03g04050D BnaA10g30020D BnaC09g44160D BnaA07g31540D BnaCnng06970D BnaA06g30710D BnaC03g65090D BnaA09g06090D BnaA02g02580D BnaA07g31680D BnaA08g10420D BnaA02g06100D BnaC01g14080D BnaC04g45680D BnaA02g10750D BnaC02g44890D BnaC09g33360D BnaA03g58600D BnaC02g04390D BnaC02g41860D BnaA09g11280D BnaC03g15450D BnaA08g26320D BnaA09g11780D BnaA09g52130D BnaCnng01390D BnaA03g17330D BnaCnng43450D BnaA03g12580D BnaCnng21270D BnaA10g18810D BnaC06g23040D BnaC07g37430D BnaA05g06890D BnaC04g07760D BnaA07g05740D BnaC03g43590D BnaA10g12770D BnaC01g01250D BnaC03g20340D BnaA09g05480D BnaC09g05060D BnaC09g36060D BnaC06g32180D BnaA01g03490D BnaA09g39030D BnaA07g30860D BnaC02g22160D BnaA01g30350D BnaA03g24010D BnaC03g28550D BnaA04g28770D BnaA08g08440D BnaC01g20650D BnaC08g48600D BnaC09g41070D BnaC08g18090D BnaA09g44500D BnaC08g37160D BnaA01g02240D BnaA07g24010D BnaC04g51450D BnaA03g37010D BnaC07g08320D BnaC05g40480D BnaAnng02150D BnaC06g08330D BnaC09g14760D BnaC08g37480D BnaA01g08670D BnaC07g34790D BnaAnng13560D BnaC02g22630D BnaA02g16690D BnaC09g50990D BnaC05g14250D BnaA06g24760D BnaCnng13370D BnaA03g10180D BnaCnng06440D BnaA07g31220D BnaA08g15280D BnaA06g12690D BnaA01g34510D BnaC01g02200D BnaC03g76670D BnaA02g26580D BnaA03g47510D BnaC07g39760D BnaA10g19370D BnaC09g54480D BnaC03g03210D BnaC03g12860D BnaC08g18080D BnaA02g17000D BnaC05g43350D BnaC02g07210D BnaCnng78680D BnaA01g30920D BnaA01g31080D BnaC05g36990D BnaA03g02860D BnaA08g22580D

Group Time BnaC03g39170D BnaA03g36940D BnaA08g27770D BnaA10g02500D BnaA10g02490D BnaC05g02370D BnaA04g27430D BnaC08g09640D Novel03836 BnaA06g02010D BnaA05g23300D BnaA01g07810D BnaC01g09350D BnaA04g13090D BnaA09g50600D Novel02250 BnaC01g09830D BnaA01g08280D BnaC07g41460D

AUX/IAA

ck_12 h_group T_12h_group ck_1 d_group T_1 d_group ck_4 d_group T_4 d_group ck_7 d_group T_7 d_group

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2 1

Group ck T

Time 12 h 1d 4d −1 7d 0

−2

Fig. 10 The differential expression patterns of transcription factors at 12 h, 1 d, 4 d, and 7 d, including AP2/ERF, AUX/ IAA, NAC, MYB, and WRKY. The ck_12 h group indicate an average gene expression level of three replicates in the ck_12 h library, and the same notation describes as T_12 h group, ck_1 d group, and T_1 d group. ck, the control; T, treatment.

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PIN3, which is transiently induced in the endodermis during the early stages of lateral root initiation, enables a proper auxin gradient for transition from FC to lateral root initiation (Marhavý et al. 2013). In our study, the endogenous IAA concentration decreased by 50 to 70% after 0.18 µmol L–1 exogenous GR24 application in comparison with the control. Moreover, the tryptophan metabolism pathway and plant hormone transduction pathway were identified by RNA transcript and metabolic profiling analysis. Therefore, we speculated that the decrease of endogenous auxin might have positive effects on the lateral root development in rapeseed. Rapeseed is an allotetraploid species, so there is the potential to create auxin or SLs mutants to further elucidate the mechanisms of gene expression in lateral root development.

50

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Down-regulated Up-regulated

40 30 20 10 0

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A

Many studies have proposed that the cross-talk between SLs, auxin and ethylene plays a role in determining root architecture (Chen et al. 2003; Kapulnik et al. 2011). SLs have been proven to affect root formation and elongation by interfering with the inhibitory effect of polar auxin transport in Arabidopsis, tomato (Solanumly copersicum), and rice (Oryza sativa) (Kotai et al. 2010; Ruyter-Spira et al. 2011; Sun et al. 2015). In addition, ethylene signaling is also involved in the strigolactone response. The ability of SLs to affect auxin flux and thereby lateral root formation in the roots, is mediated by the induction of ethylene synthesis (Koltai 2011a). In our study, GO functional enrichment analysis indicated that “metabolic process” and “response to stimulus” were over-represented terms for DEGs. Moreover, the results showed that the metabolic, phenylpropanoid

350

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a a

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Fig. 11 The differential metabolites and endogenous indole-3-acetic acid (IAA) concentrations at the four time points. A, comparison between the metabolites in the lateral root tissues after GR24 treatment (T) with that in the control (ck) at 12 h, 1 d, 4 d, and 7 d. Metabolites with significant differences in content were set with thresholds of variable importance as VIP value>1, fold change≥2 or fold change≤0.5. B, the endogenous IAA concentration in the lateral root tissues of treated plants and ck at the four time points. Letters mean significant differences (P-value<0.05) and bars mean standard errors of replications triplicate.

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C Bn 04g aC 01 8 0 Bn 9g 10D aA 39 6 0 Bn 5g 80D 3 aA 48 0 Bn 3g 50D aA 17 0 1 Bn 0g 00D 1 aA 66 Bn 01g 60D aC 23 1 Bn 01g 20D aC 42 8 0 Bn 8g 90D aA 36 6 0 Bn 1g 40D 0 aA 23 Bn 07g 90D aC 24 8 Bn 06g 90D aC 20 02 69 g0 0D 23 50 D

0

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Fig. 12 Quantitative real-time PCR (qPCR) validations of gene expression levels from differential gene expression (DGE) analysis. Three candidate genes at each time point were randomly chosen for verification. The bars mean standard errors of triplicate.

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biosynthesis, and tryptophan metabolism pathways played important roles in the regulation of lateral root growth after GR24 treatment. Phenylpropanoid pathway is responsible for the synthesis of numerous compounds important for plant growth and responses to the environment. Tryptophan is the precursor of the IAA. The interaction of auxin and phenylpropanoid impacts both shoot and root development, and auxin regulates plant growth is fine-tuned by early steps in phenylpropanoid biosynthesis (Kurepa et al. 2018). Transcriptome analysis reveals interplay between hormones, ROS metabolism and cell wall biosynthesis for drought-induced root growth in wheat. Drought stress not only significantly up-regulated auxin receptor (AFB2), but also up-regulated ACC oxidase and 2OG-Fe(II) oxygenase which might contribute to maintenance of a basal ethylene level to maintain root growth (Dalal et al. 2018). The present study reports AXR3, PBS3 and ERF2, the auxin and ethylene responsive family proteins assigned to hormone signal transduction, were identified by KEGG analysis. Although the mechanism of the relationship between SLs, ethylene and auxin signaling is unclear, these results suggest that the cross-talk junction between SLs, auxin and ethylene is very relevant in regulating lateral root formation in rapeseed. Overall, the abundance of genes assigned to hormone signal transduction, the hormone biosynthesis pathway and hormone responsive proteins might have great implications for lateral root development in rapeseed.

4.3. Lateral root development regulation by reactive oxygen species (ROS) generation and transcription factors The ability of plants to survive the constantly changing environment relies upon a network of sophisticated mechanisms to perceive and properly respond to their surroundings (Stepanova et al. 2008). Zhang et al. (2014) has suggested that plant hormones modulate lateral root development in an indirect way, mostly by regulating the ROS generation and transcription factors. ROS generation adjusts the membrane system, modifies the cell wall architecture and changes cell division (Opitz et al. 2015). Lipids act as signaling molecule to maintain cell membrane properties. Lipid metabolism (glycerolipid metabolism/ glycerophospholipid metabolism) was predominant at four time points in our study. Additionally, ROS plays a central role as signaling molecules, and spatial and temporal accumulation has strong impacts on hormone synthesis, transport, localization, and signaling (Mittler et al. 2011). Genes encoding the enzymes for detoxifying ROS, such as catalases, supeoxide dismutases, and peroxidases, were particularly over-represented in the elongation zone and

cortex (Opitz et al. 2015). Here, GO analysis enriched the terms of “catalytic activity”, “response to stress stimulus”, and “oxidoreductase activity” at the four time points, which were in agreement with previous studies (Zhang et al. 2014; Opitz et al. 2015). Peroxidases are particularly abundant in roots (Dunand et al. 2007). Peroxidase superfamily proteins including PRX52, Per4, and others, were identified among the transcripts that were differentially expressed when treated with GR24, and which might play a role in generating H2O2 during the defense response. Further study revealed that some of the peroxidases were involved in the phenylpropanoid biosynthesis pathway, which was confirmed by RNA transcript and metabolic profiling conjoint analysis. The MAPK signaling pathways are common and versatile signaling components that lie downstream of second messengers and hormonal (Xu and Zhang 2015). Available data have shown that MAPK cascades play central roles in plant responses to pathogens, drought, salinity, cold, wounding, ozone, ROS, and hormone stimuli (Berriri et al. 2012; Raja et al. 2017). In this study, the MAPK signaling pathways were enriched in response to GR24 treatments that affected lateral root growth. Based on our analysis, we highlight the link between ROS and the GR24 induced lateral root formation in rapeseed. A multitude of transcription factors including AP2/ERF, AUX/IAA, bZIP, bHLH, LOB, GARP, HSF, MADS, WRKY, NAC, and MYB, were over-represented in GR24 treated root tissues. The AP2/ERF family is one of the major plant transcriptional regulators participating in plant growth and development (Nakano et al. 2006; Jin et al. 2018). Our study demonstrated that the ethylene responsive transcription factors ERF003 and ERF107 were significantly up-regulated after GR24 treatment. ARF7 and ARF19 control lateral root organogenesis via auxin dependent degradation of AUX/ IAAs, such as IAA14 and IAA19 (Tatematsu et al. 2004; Okushima et al. 2007; Cho et al. 2014). In the present study, some primary auxin response genes such as IAA18 and IAA26, were clearly up-regulated. The WRKY transcription factors can function as either positive or negative regulators of gene expression, which have been proven to regulate the cell wall loosening and lateral root growth (Zhang et al. 2014). WRKY33 was remarkably up-regulated after GR24 treatment in our study as reported by Zhang et al. (2014). NACs belong to a plant-specific family that plays important roles in many aspects of plant growth and abiotic stress responses (Hao et al. 2011; Nakashima et al. 2012; Escobar-Sepúlveda et al. 2017). NAC29 and NAC55 are transcription factors that may be involved in translating the environmental and endogenous stimuli into the process of plant lateral root development, and the expression level of these genes are induced by auxin signaling (Zhang et al.

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2014). In the current study, NAC29, NAC55, and NAC100 were identified. The MYB transcription factors comprise one of the largest transcription factor families in the Arabidopsis genome, which can regulate lateral root meristem activation and both positively and negatively regulate the stress tolerance of plants (Seo and Park 2009; Gao et al. 2016). For instance, MYB96 is a molecular link that integrates ABA and auxin signals in modulating auxin homeostasis during lateral root development (Seo and Park 2009). As described above, GO terms of “response to stress stimulus” and “oxidoreductase activity” were enriched, and some primary auxin response genes were identified. Therefore, it is possible that the plant responses to stress, ROS, and hormone stimuli are activated after GR24 treatment and mostly MYB transcription factors are up-regulated at the 12 h and 1 d.

5. Conclusion GR24 treatment promoted the plant growth of rapeseed that involved increasing the root-shoot dry weight, root tips, and lateral root length. Transcriptomics analysis showed that the DEGs in the lateral root tissues between GR24 treatment and control in the lateral root tissue were involved in plant hormone transduction, tryptophan metabolism pathway, and phenylpropanoid biosynthesis pathway. Furthermore, the transcription factors including AP2/ERF, AUX/IAA, bZIP, bHLH, LOB, GARP, HSF, MADS, WRKY, NAC, and MYB were significantly differentially expressed. Interestingly, metabolomics analysis demonstrated that the concentrations of endogenous IAA in lateral root tissue decreased significantly after the GR24 treatments. In summary, our study suggests that low concentrations of GR24 promote the lateral root development mediated by plant hormones, i.e., auxin and ethylene signaling. The data obtained in this study could contribute to further illuminating the molecular mechanisms of lateral root development in response to GR24 treatment in rapeseed and lay the foundation for improving crop productivity through plant hormone regulation.

Acknowledgements We express our thanks to Prof. Wang Hanzhong (Chinese Academy of Agricultural Sciences) for providing the seeds of Zhongshuang 11 (ZS11). Funds were provided by the National Key Research and Development Program of China (2018YFD1000900). Appendices associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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Executive Editor-in-Chief ZHANG Xue-yong Managing editor WANG Ning