Genome-wide characterization of BnaLEA gene family reveals a positive role of BnaLEA39 in freezing tolerance in rapeseed (Brassica napus L.)

doi:10.21203/rs.3.rs-3904496/v1

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Research Article

Genome-wide characterization of BnaLEA gene family reveals a positive role of BnaLEA39 in freezing tolerance in rapeseed (Brassica napus L.)

https://doi.org/10.21203/rs.3.rs-3904496/v1

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Background

Freezing stress is one of the major abiotic stresses that causes extensive damage to plants. LEA (Late embryogenesis abundant) proteins play a crucial role in plant growth, development, and abiotic stress. However, there is limited research on the function of LEA genes in low-temperature stress in Brassica napus (rapeseed).

Results

Total 148 potential LEA genes were identified in B. napus and divided into eight subgroups. BnaLEA genes of the same subgroup had similar gene structures and predicted subcellular locations. Cis-regulatory elements analysis showed that the promoters of BnaLEA genes rich in cis-regulatory elements related to various abiotic stresses. Additionally, RNA-seq and real-time PCR results indicated that the majority of BnaLEA family members were highly expressed in senescent tissues of rapeseed, especially during late stages of seed maturation, and most BnaLEA genes can be induced by salt and osmotic stress. Interestingly, the BnaLEA39 and BnaLEA40 genes were highly expressed across different vegetative and reproductive organs during different development stages, and showed strong responses to salt, osmotic, and cold stress, particularly freezing stress. Further analysis showed that overexpression of BnaLEA39 increased the freezing tolerance in rapeseed, as evidenced by lower relative electrical leakage and higher survival rates compared to the wild-type (WT) under freezing treatment.

Conclusion

This study is of great significance for understanding the functions of BnaLEA genes in freezing tolerance in rapeseed and offers an ideal candidate gene (BnaLEA39) for molecular breeding of freezing-tolerant rapeseed cultivars.

Brassica napus

LEA (Late embryogenesis abundant)

freezing stress

abiotic stress

Plants, as sessile organisms, are susceptible to abiotic stresses throughout their growth and development. Due to global climate change, the frequent occurrence of extreme weather events has increased the risk of low-temperature stress in plants, which in turn severely affects crop yields[1, 2]. Plant cell membranes are most susceptible to cold damage[3–5]. Chilling temperatures (above 0 ℃) have impact on the fluidity of cell membranes and thus hindering plant growth and development, while freezing temperature (below 0 ℃) result in the disruption of cell membranes, ultimately leading cell death[6]. Plants have evolved various adaptations at the molecular, cellular, and physiological level to cope with different growth conditions, one such adaption involves the expression of a specific class of proteins known as late embryogenesis abundant (LEA) proteins[7–10].

LEA proteins are a class of proteins characterized by their high hydrophilicity, thermal stability, and intrinsic disorder. Most LEA proteins contain hydrophilic amino acids such as glycine, lysine, and histidine[11]. Based on sequence similarity and protein family domains (Pfam), LEA proteins can be divided into eight subgroups: LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, DHN (Dehydrin), and SMP (Seed Maturation Protein)[12]. LEA proteins were first discovered in the late stages of cotton seed maturation and have since been found in roots, stems, leaves, and flowers of plants[13, 14]. It has been shown that LEA proteins can maintain basic cellular metabolism by adsorbing water molecules under dehydration conditions and stabilize membranes by binding to them[15, 16]. Additionally, LEA proteins play important roles in maintaining the stability of the cellular matrix in a glassy state, ionic binding, molecular protection, and preservation of enzyme activity and double-stranded DNA binding[17–19]. These characteristics make LEA proteins play important role in protecting plants from abiotic stress conditions. Overexpression of a cucumber Y3SK2 type DHN gene CsLEA11 in E. coli enhanced cell viability and conferred heat and cold tolerance[20]. Overexpression of maize ZmLEA3 enabled tobacco, yeast, and E. coli to tolerate low-temperature stress[21]. Tomato strains expressing SiLEA4 had significantly greater freezing resistance, due to a significant increase in the antioxidize activities and proline content[22]. Both LEA3 from rapeseed and Arabidopsis primarily improve drought tolerance and oil content by enhancing photosynthetic efficiency and reducing reactive oxygen species (ROS) accumulation[23]. Linkage of LEA genes in low temperature tolerance during seed germination in B. napus by weighted gene co-expression association transcriptional regulatory network analysis[24]. Overexpression of the BcLEA73 gene in Arabidopsis has been found to significantly enhance their tolerance to salt, drought, osmotic stress, and low temperature[25].

Rapeseed (Brassica napus: genome AnAnCnCn) is an important oil-seed crop, which was formed ~ 7500 years ago by allopolyploid between the ancestor of B. rape (genome ArAr) and B. oleracea (genome CoCo)[26, 27]. The growth and development of rapeseed are severely impacted by environmental conditions. Although there were some studies about the functions of LEA genes in salt and drought tolerance, seed oil content in rapeseed, the research about their functions in freezing tolerance in rapeseed remains limited. In this study, 148 putative LEA genes were identified in B. napus, and their structural features, chromosome localization, evolutionary relationship, promoter sequence, and expression patterns in various tissues and under different abiotic stresses were evaluated. Additionally, we confirmed that overexpression of the BnaLEA39 gene enhances the freezing tolerance in rapeseed seedlings. This study will provide valuable information for deciphering the biological functions of BnaLEA genes in the response of freezing stress in rapeseed.

Identification of BnaLEA genes in B. napus

The protein, CDS, and DNA sequences of 51 Arabidopsis AtLEAs were downloaded from TAIR10 in Ensembl gemones (http://ensemblgenomes.org/). Whole-gene data of Brassica napus was downloaded from Brassica napus pan-genome information resource (http://cbi.hzau.edu.cn/bnapus/)[28]. Hidden Mark Model profiles of LEA proteins with accession numbers PF03760 (LEA_1), PF03168 (LEA_2), PF03242 (LEA_3), PF02987 (LEA_4), PF00477 (LEA_5), PF10714 (LEA_6), PF00257 (DHN), and PF04927 (SMP) downloaded from the Pfam database (https://pfam.xfam.org/), were used to HMM search against the local genome database of B. napus using TBtoolsv.120[29]. Fifty-one AtLEA proteins were used as query sequences for the search of B. napus LEA proteins by BLASTP (E-value < 1e^− 5) in TBtoolsv.120[29]. All members were verified for the presence of LEA repeats using Conserved Domain (CD)-search in NCBI (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). The subcellular localizations of LEAs were predicted using ProtComp 9.0 (http://linux1.softberry.com/). The molecular weight (kDa), theoretical isoelectric point (pI) and grand average of hydropathy (GRAVY) of BnaLEAs were predicted by EXPASY (https://web.expasy.org/protparam/)[30]. The TBtoolsv1.120[29] was used to analyze the gene structure of LEAs. The conserved motifs were analyzed with MEME program (http://meme.nbcr.net/meme/cgi-bin/meme/cgi)[31].

Phylogenetic analysis of the LEA proteins

The sequence of LEA proteins was aligned using Muscle, and a phylogenetic tree was constructed using the Maximum-likelihood (ML) phylogenetic method with 1000 bootstrap replicates in TBtoolsv.120[29].

Prediction of cis-regulatory elements in promoter sequences of BnaLEA genes The 2 kb promotor region of each BnaLEA gene was submitted to the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) promoter analysis tool to identify potential cis-regulatory elements.

Expression analysis of BnaLEA genes

To analyze the expression pattern of BnaLEA genes in various tissues as well as under phytohormonal and abiotic stresses of B. napus, the publicly available RNA-seq dataset of ZS11 (semi-winter type B. napus cv. Zhongshuang 11) were retrieved from BnIR (Brassica napus multi-omics information resource https://yanglab.hzau.edu.cn/BnIR)[28]. Gene expression levels were calculated using the TPM method (transcripts per million), and the expression values of the BnaLEA gene were shown in the Table S3. The log₂(TPM + 1) value of the BnaLEA gene was used to generate heat map by TBtoolsv1.120[29]. qRT-PCR analysis of BnaLEA39 was performed as described previously[32–34]. The primers used in this study were listed in Table S4.

Obtain of BnaLEA39 overexpression rapeseed lines and phenotype assay

In this paper, the semi-winter rapeseed variety ZhongShuang-6 was obtained commercially from Wuhan Towin Biotechnology Co., Ltd., Wuhan, China. We constructed and transferred the 35S overexpression vector of BnaLEA39 to ZhongShuang-6. The relative expression level of BnaLEA39 gene in BnaLEA39-overexpression lines and wild-type (WT) were analyzed by qRT-PCR. Two BnaLEA39-overexpression lines (OE3, and OE4) were selected for subsequent experiments. BnaLEA39-overexpression lines and WT were seeded in nutrient soil and placed in an artificial climate chamber for growth. The growth conditions were as described previously[35]. The 30-day-old seedlings of WT and BnaLEA39-overexpression lines were subjected to freezing treatments. The freezing treatment was carried out by reducing the temperature from 20 ℃ to 0 ℃, and then reduced to -3.5 ℃ in a gradient of 1 ℃ per hour, with the − 3.5°C lasting for 2 hours, followed by recovery at 20 ℃ for 7 days. The phenotypic change of B. napus were photographed, and the survival rate was counted. The relative electrical conductivity contents were detected by the method according to provious study[36]. Three replicates were performed for each sample.

Statistical Analysis

All results in this study were performed in more than three replicates. Data were expressed as mean of triplicate values, and the error represented the SEM (Standard Error of Mean). Statistical analysis and plotting were performed using GraphPad Prism8 (V8.4.3, GraphPad, Changchun, China). The statistical significance of difference was confirmed by one-way ANOVA.

Genome-wide identification of BnaLEA in rapeseed

A total of 148 LEA proteins were identified in the B. napus based on the BLASTP results of AtLEAs protein sequence and Hidden Mark Model, which were named as BnaLEA1 to BnaLEA148, respectively (Table.S1). Based on their conserved domain structures, the BnaLEA proteins were classified into eight families. Most proteins in the same subgroup had similar parameters. The 148 BnaLEA proteins encoded 57–298 amino acids (AA) with the CDS ranging from 174 bp (BnaLEA140) to 897 bp (BnaLEA19). The predicted molecular weights (Mw) of these BnaLEA proteins ranged from 6.61 KDa (BnaLEA140) to 33.66 KDa (BnaLEA19). In general, LEA proteins are relatively small, but some larger LEA proteins (~ 79.20 kDa) were found in the LEA_4 subgroup, which was consistent with the research on LEA proteins in Arabidopsis[37]. Approximately two-thirds of LEA proteins had relatively low isoelectric points (pI < 7.0), including the SMP, DHN, LEA_2, LEA_5 and LEA_6 subgroups, the remaining proteins, particularly those in LEA_1 and LEA_3 subgroups, had pI > 7.0. The calculated grand range average of hydropathy index (GRAVY) values of all BnaLEAs ranged from − 1.893 to 0.093. GRAVY values of 141 BnaLEA proteins (95%) were below 0, suggesting that most of the BnaLEAs were hydrophilic. The predicted subcellular localizations of these proteins revealed that most of LEA proteins may be in nucleus (100 members), while few members were in mitochondrion (13), plasma membrane (14), chloroplast (3) or extracellular space (18).

Phylogenetic analysis

A phylogenetic tree (Fig. 1) was generated using the sequences of 199 LEA proteins from B. napus (148) and Arabidopsis (51). Consistent with previous studies[38], the LEA proteins were divided into eight groups. The LEA_4 subgroup was the largest, with 52 members which were mainly clustered in 3 branches. The LEAs of LEA_2 was clustered into 2 branches and the LEAs of LEA_1, LEA_3, LEA_5, LEA_6, SMP and DHN were clustered into a separate branch. However, AT3G17520, which contained a LEA_4 domain, clustered into another clade, closer to the LEA_6 family. All 51 AtLEAs were found to have orthologous genes in rapeseed, and most of them had only one to five orthologous genes. There was an expansion of three AtLEAs (AT4G13560, AT3G22490/AT3G22500, and AT4G02380) in rapeseed and a complete duplication of three AtLEAs (AT2G46140, AT5G44310, and AT1G20450/AT1G20440) had six orthologous genes in rapeseed.

Motif analysis and gene structure analysis of LEAs

To explore the conserved LEAs motifs, five motifs were identified using MEME (Fig. S1). The result showed that the distribution patterns of these five motifs were similar in LEA members from same subgroup, such as LEA_1 (motif-2-1-4-3-5), LEA_5 (motif-2-5-3-1-4), DHN (motif-4-3-2-1-5), and SMP (motif-4-3-1-2-5), whereas in other subgroups, such as LEA_2, LEA_4, and LEA_6, although the distribution patterns of motifs varied among the different members, the distribution of AtLEAs and their orthologous BnaLEAs were similar.

To investigate the structural characteristics of BnaLEA genes, the exon-intron structure (Fig.S2 and Table S1) of BnaLEA genes were analyzed. The results showed that all LEA_6 subgroup genes had no intron (except BnaLEA36), all LEA_5 subgroup genes and most of LEA_1, LEA_3 and DHN subgroup genes had 2 exons, LEA_2, LEA_4 and SMP genes had 1 to 4 exons.

Cis -regulatory elements analysis of the BanLEA promotors

To understand the potential regulatory mechanisms of BnaLEAs, cis-regulatory elements were analyzed using PlantCARE. A total of 4097 elements were predicted in the promoters of 148 BnaLEAs. There are 1550, 2244, 120 and 183 cis-regulatory elements related to plant hormone response, environmental stress response, organ development, and transcription-factor binding sites, respectively (Fig. S3 and Table S2). Among them, the light-responsive, ABA-responsive, and MeJA-responsive cis-regulatory elements were the top three. The result indicated that most BnaLEAs could be regulated by various plant hormones and environmental stresses in rapeseed.

Expression profiling of the BnaLEA genes in different tissues and under abiotic

stress and phytohormone treatments

To investigate the expression patterns of the BnaLEA genes, their expression patterns in five different tissues/organs of rapeseed (root, stem, leaf, seed, and silique), under stresses (salt, drought, freezing, cold, heat, and osmotic), and phytohormone treatments (indole-3-acetic acid, IAA; 1-aminocyclopropane-1-carboxylic acid, ACC; gibberellic acid, GA; abscisic acid, ABA; cytokinin, TZ; jasmonate, JA; and brassinolide, BL) were determined by analyzing RNA-Seq data (Table S3)[39]. Most of the BnaLEA genes (78/148) were highly expressed in mature seeds and lowly expressed in other tested tissues (Fig. 2), which is consistent with the characterization of LEA genes that accumulate in large amounts during the late stage of seed development[40]. Unexpectedly, most of the LEA_2, LEA_3 and DHN subgroup members, as well as BnaLEA39 and BnaLEA40 from LEA_6, were highly expressed across different vegetative and reproductive organs during different development stages (Fig. 2).

Members of the BnaLEA gene family exhibited similar expression patterns under different stresses in roots and leaves (Fig. 3, and Fig S4). The expression of most BnaLEA genes was up-regulated by salt stress, freezing stress, and osmotic stress. Additionally, some genes were particularly responsive to freezing stress. Examples included some members from the LEA_4 subgroup (BnaLEA57, BnaLEA58, and BnaLEA59), and the LEA_6 subgroup (BnaLEA39 and BnaLEA40). Less predictably, most LEA subgroup members showed slightly or no response to hormones-treatments in leaves (99/148) and roots (76/148) (Fig. 4 and Fig S5). However, a few LEA subgroup members were strongly up-regulated induced by different hormones-treatments (Fold change > 2 and TPM > 1), and they exhibited distinct expression patterns in leaves and roots. Specifically, in leaves, 5, 2, 5, 10, 0, 4, 14 and 1 members of the LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, DHN, and SMP subgroups, respectively, exhibited up-regulation in response to various hormone-treatments. conversely, in roots, 3, 2, 6, 19, 3, 5, 17, and 4 members of the LEA_1, LEA_2, LEA_3, LEA_4, LEA_5, LEA_6, DHN, and SMP subgroups, respectively, were up-regulated by hormone-treatments, with expression levels were generally higher in roots than in leaves. Notably, the LEA genes demonstrated a more pronounced response to ABA both in leaves and roots (Fig. 4 and Fig S5).

To validate the results of transcriptome data, we performed a qRT-PCR to detect the transcript levels of BnaLEA39 gene in different tissues (root, stem, leaf, and seed) of rapeseed and under freezing stress treatments (Fig. 5a, b). The BnaLEA39 gene was expressed in the roots, stems, and leaves of the plant, especially exhibiting high expression levels during 40 day-old seeds (Fig. 5a). Additionally, the expression of the BnaLEA39 gene was significantly up-regulated under freezing stress, with or without cold acclimation (Fig. 5b). These results implied that the BnaLEA39 gene may plays an important role in the growth, development, and response to freezing stress in rapeseed.

Overexpression of BnaLEA39 enhances freezing tolerance in rapeseed

To further investigate the function of the BnaLEA39 gene under freezing stress, we constructed a 35S:BnaLEA39 vector and obtained six BnaLEA39 overexpression lines in Zhongshuang-6. Among them, two lines (OE4 and OE5) with the highest expression were selected for freezing treatment (Fig. 6a, b). After freezing treatment at -3.5 ℃ for 2 hours, the BnaLEA39 overexpression lines showed only partial leaf icing and water-soaking, while most of the WT exhibited severe icing and water-soaking throughout the whole plant, accompanied by plant softening (Fig. 6a). Additionally, the relative electrical conductivity of the overexpression lines was significantly lower than that of the WT (Fig. 6c). Following seven days of recovery, the BnaLEA39 overexpression plants showed significantly better growth and higher survival rates compared to the WT plants (Fig. 6a, d). These results indicate that BnaLEA39 can enhance the freezing tolerance of rapeseed.

Low temperatures have detrimental effects on plant growth and even result in plant death. It is imperative to explore genes that enhance the low-temperature tolerance in rapeseed. Numerous studies have shown the positive impact of LEA gene expression on stress tolerance in several crops, such as rice[40], maize[41], wheat[42], and rapeseed[43]. Nevertheless, there is a scarcity of reports specifically focusing on the role of LEA genes in conferring freezing tolerance in rapeseed.

In this study, 148 LEA genes were identified in rapeseed and can be divided into eight subgroups (Fig. 1). The LEA_4 subgroup having the highest number of members, consistent with previous studies in Arabidopsis[44] and B. napus[38]. Despite the limited sequence similarity among members within the LEA_4 subgroup, as indicated by their distribution across multiple branches, they all share the characteristic Pfam structural domain (Table S1 and Fig. 1). Previous studies in Arabidopsis and rice have shown that multi-stimuli response genes were shorter and had fewer introns[45, 46]. 72.2% (107/148) of BnaLEA genes contained 0–1 introns, and all members of the LEA_6 subgroup (except BnaLEA36) have no introns (Table S1 and Figure S1). Correspondingly, the promoter sequences of LEA_6 members were rich in light-responsive and defense hormone-responsive cis-regulatory elements, and LEA_6 members were induced by abiotic stress and hormones-treatments (Table S2 and Figure S3).

Typical LEA genes were exhibited high expression levels in the late stages of seed development in Arabidopsis[44] and rice[40], Similarly, we found that most of the BnaLEA gene family members are also highly expressed in the late stage of seed development (Table S3 and Fig. 2). However, there also existed a portion of genes, such as BnaLEA5 and BnaLEA6 from LEA_2 subgroup, and 10 members from DHN subgroup, were instead highly expressed in the early stages of seed development and other tissues (Table S3 and Fig. 2), which suggested the diversity functions of those LEAs. Transcriptome data suggested that a few BnaLEAs were induced by multiple hormones in leaves and roots, especially ABA. Additionally, most of those genes had a broader, earlier, and stronger response in roots under hormone treatments than that in leaves (Table S3, Fig. 4 and Figure S5). Notably, BnaLEA39 and BnaLEA40 were differed with the other members from LEA_6 subgroup in being induced by IAA, ACC, and ABA hormones in leaves, and by ABA only after 6 h in roots. ABA is widely involved in plant response to adversity[47, 48]. This implied that BnaLEA39 and BnaLEA40 played a unique role in multiple stress tolerances in rapeseed.

It can obtain clues form gene expression patterns to explore the function of genes[49]. Previous studies had shown that overexpression of the BnLEA57 (named BnaLEA98/BnaC05G0444500ZS in this study) gene or its homolog BnLEA55 (named BnaLEA97/BnaA05G0396800ZS in this study) in Arabidopsis increased seed oil content[50]. In rice, the overexpression of OsEm1, which encoded a group I LEA protein, had been shown to increase the survival rate of rice plants under drought stress during nutritional stages[51]. MsLEA (LEA_4, AT4G13230) recruited and protected its target proteins (SOD and Ms1770) and increased alfalfa tolerance against drought and aluminum stresses[52]. However, the enhancement of plant low temperature tolerance through LEA gene is relatively limited. Interestingly, expression analysis revealed that BnaLEA39 gene was not only highly expressed during seed development, but also highly expressed in nutrient organs across various developmental stages of the plant (Fig. 2, and Fig. 5a). Therefore, we speculated that the BnaLEA39 gene was associated with plant growth and development and oil content in rapeseed. Additionally, the BnaLEA39 gene showed significant upregulation in freezing treatment, with or without cold acclimation (Fig. 5b), implying that the BnaLEA39 gene may has significant functions in the freezing tolerance. Further research found that the BnaLEA39-overexpression rapeseed lines shown higher freezing tolerance than WT, coupled with lower relative electrical rate and higher survival rate under freezing stress conditions.

In this study, 148 BnaLEA genes were identified in B. napus, which were categorized into eight subgroups. 72.2% (107/148) of BnaLEA genes contained 0–1 introns. Most cis-regulatory elements in the promotors of BnaLEA were related to plant hormone and environmental stress response. BnaLEA genes displayed different spatiotemporal expression patterns, as well as various abiotic stress and hormone responsive expression patterns. Most of them were up-regulated induced by salt stress, freezing stress, and osmotic stress. Overexpression of BnaLEA39 resulted in an increased freezing tolerance in rapeseed. This study contributes to our understanding of LEA genes in rapeseed and offer an ideal candidate gene (BnaLEA39) for molecular breeding of freezing-tolerant rapeseed cultivars.

Ethics approval and consent to participate

All the methods involving plants and their materials complied with relevant institutional, national, and international guidelines and legislation.

Consent for publication (Not applicable)

Availability of data and materials

The data that support this study are available in the article and accompanying online supplementary material.

Competing interests

The authors report no declarations of interest.

Funding

This work was supported by the Scientific and Technological Innovation 2030 [grant number 2022ZD0400901] and Science and Technology Innovation Program of Hunan Province [grant numbers 2020RC2057].

Authors’ contributions

Weiping Wang: Formal analysis, Methodology, Writing-original draft. Yan Liu: Data curation, Formal analysis. Yu Kang: Data curation, Formal analysis. Wei Liu: Data curation, Formal analysis. Shun Li: Data curation, Formal analysis. Zhonghua Wang: Data curation, Formal analysis. Xiaoyan Xia: Data curation, Formal analysis. Xiaoyu Chen: Data curation, Formal analysis. Lunwen Qian, Supervision. Xinghua Xiong, Supervision. Zhongsong Liu, Supervision. Chunyun Guan, Supervision. Xin He: Conceptualization, Funding acquisition, Writing-review & editing.

Acknowledgements

Not applicable.

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No competing interests reported.

Supplementaryfigure1.pdf
Supplementary figure 1: MEME analysis of LEA proteins. Five motifs in Arabidopsis and B. napus LEA proteins were identified by MEME and were represented by five boxes of different colors.
Supplementaryfigure2.pdf
Supplementary figure 2: Gene structures of BnaLEA genes analyzed by TBtools. Orange boxes, black lines, and green boxes indicate untranslated regions, introns, exons, respectively.
Supplementaryfigure3.pdf
Supplementary figure 3: The cis-regulatory elements in the promoters of BnaLEAs predicted by PlantCARE. Boxes filled with different colors represent different cis-regulatory elements.
Supplementaryfigure4.pdf
Supplementary figure 4: Expression of BnaLEA genes under different abiotic stresses in roots. Colored rectangles indicate expression levels of BnaLEA genes. Red means high expression, Blue means low expression. Color from red to blue represents descending log₂(TPM+1).
Supplementaryfigure5.pdf
Supplementary figure 5: Expression of BnaLEA genes under hormones treatments in roots. Colored rectangles indicate expression levels of BnaLEA genes. Red means high expression, Blue means low expression. Color from red to blue represents descending log₂(TPM+1).
Supplementarytable1.xlsx
Supplementary table 1: Characteristics of BnaLEAs in B. napus.
Supplementarytable2.xlsx
Supplementary table 2: Cis-regulatory elements in promotors of BnaLEAs.
Supplementarytable3.xlsx
Supplementary table 3: RNA-seq data of expression levels of BnaLEAs in the different tissues and under different abiotic stresses and plant hormone treatments in leaves and roots.
Supplementarytable4.xlsx
Supplementary table 4: List of primers used in this study.

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Genome-wide characterization of BnaLEA gene family reveals a positive role of BnaLEA39 in freezing tolerance in rapeseed (Brassica napus L.)

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Abstract

Background

Results

Conclusion

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Introduction

Materials and methods

Phylogenetic analysis of the LEA proteins

Statistical Analysis

Results

Genome-wide identification of BnaLEA in rapeseed

Phylogenetic analysis

Motif analysis and gene structure analysis of LEAs

stress and phytohormone treatments

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1