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. 2023 Oct 13;18(10):e0292898. doi: 10.1371/journal.pone.0292898

Identification and characterization of lipoxygenase (LOX) genes involved in abiotic stresses in yellow horn

Fang Hu 1, Yunxiang Zhang 2, Jinping Guo 1,2,*
Editor: Mojtaba Kordrostami3
PMCID: PMC10575502  PMID: 37831731

Abstract

Lipoxygenase (LOX) gene plays an essential role in plant growth, development, and stress response. 15 LOX genes were identified, which were unevenly distributed on chromosomes and divided into three subclasses in this study. In promoter region analysis, many cis-elements were identified in growth and development, abiotic stress response, hormonal response, and light response. qRT-PCR showed that the LOX gene showed tissue specificity in seven tissues, especially XsLOX1, 3, and 7 were relatively highly expressed in roots, stems, and axillary buds. The different expression patterns of LOX genes in response to abiotic stress and hormone treatment indicate that different XsLOX genes have different reactions to these stresses and play diversified roles. This study improves our understanding of the mechanism of LOX regulation in plant growth, development, and stress and lays a foundation for further analysis of biological functions.

Introduction

Lipoxygenases (LOXs) are widespread in plants and animals [1], catalyzing the oxidation of polyunsaturated fatty acids (PUFA) in complex eukaryotes [2]. Based on sequence similarity, the 13-LOXs can be further partitioned into two divisions: Type I and Type II [3]. LOXs encompass two significant domains, one at the C-terminal lipoxygenase domain and another at the N-terminal polycystic protein-1, lipoxygenase, α-toxin (PLAT) domain [4].

With sequencing and functional genomics progress, studies on LOX-encoding genes are burgeoning in various plant species. Numerous previous studies have shown the function of LOXs in signaling transduction, development, and aging in plants [3, 5–8], particularly in the face of biological [7, 9–11] and environmental stresses [12–21]. Notably, AtLOX1 and AtLOX5 are associated with lateral root development [22]; AtLOX2, AtLOX3, AtLOX4, and AtLOX6 are implicated in JA biosynthesis. Moreover, AtLOX2 can be induced in response to damage. AtLOX6 is involved in drought tolerance [17], while AtLOX3 and AtLOX4 have been implicated in flower development [23]. The peanut lipoxygenase AhLOX29 [20] and oriental melon CmLOX10 [24] play a role in drought tolerance, the pepper lipoxygenase CaLOX1 [25] and the rice OsLOX10 [26] affect and resistance to high salinity stress. The persimmon DkLOX3 [18] and passiflora edulis PeLOX4 [27] promoted ripening.

Yellow horn (Xanthoceras sorbifolium Bunge) is an oil-rich seed shrub with important economic value. Due to its strong adaptability to various abiotic stress conditions such as saline-alkali environments, deserts, and arid areas, it can help to eliminate desertification and erosion and be planted as an ornamental tree. However, the systematic identification and classification of the LOX genes in yellow horn have yet to be reported. They are following the accomplishment of genome-wide sequencing, which makes it possible to pinpoint and analyze the LOX family members in yellow horn. Fifteen LOX genes have thus been identified, and each has undergone an analysis of its phylogenetic relationships, chromosomal distribution, homology, gene structure, subcellular localization, conserved protein motifs, cis-acting elements, and expression patterns. These studies provide novel insights into the exploration of essential functional genes for regulating plant growth, development, and responding to stresses in yellow horn.

Materials and methods

Identification and classification

We downloaded Xanthoceras sorbifolium genome data from the GigaScience GigaDB repository. The protein sequences of LOX in Arabidopsis thaliana, Populus trichocarp, and Oryza sativa were retrieved from the Phytozome website (https://phytozome.jgi.doe.gov/). We submitted the protein sequences as queries in the BLAST search to identify members of the gene family. A Hidden Markov Model (HMM) profile (PF00305) was obtained from the Pfam database for gene family identification with HMMER3.0. An E-value cutoff was 0.001, and default parameters were taken. Afterward, the conserved domains of candidate genes were confirmed via the NCBI Database (https://www.NCBI.nlm.nih.gov/cdd) to predict the conserved functional domains. The computing tool PI/MW of ExPASy (https://web.expasy.org/compute_pi/) was utilized to determine the PI and MW of soybean LOXs. We used WoLF PSORT online tool to predict the subcellular localization of LOX genes.

Phylogenetic analysis

The clustalW program produced alignments among various XsLOX candidates applying the default parameters [28]. Phylogenetic trees were built according to the maximum likelihood method using MEGA 7.0 [29]. Nodal support was assessed with 1,000 bootstrap replicates, and protein sequences were classified into their respective 9- and 13-LOX categories. Subsequently, the phylogenetic tree was visualized in iTOL.

Gene structure and conserved motif

The conserved protein sequence motifs of the XsLOXs were analyzed using Multiple Em for Motif Elicitation web tool (https://meme-suite.org/meme/tools/meme) with the parameters maximum of 10 motifs and a width ranging from 6 and 50 amino acid residues [30]. The structures of genes were mapped through the online Gene Structure Display Server [31], while the phylogenetic tree and conserved motifs were integrated and visualized via Tbtools [32].

Chromosome distribution and gene duplication

The physical location information of all XsLOX genes was determined from the yellow horn genomic database. We investigated collinearity relationships and gene duplication events through the MCScanX (Multiple Collinear Scanning Toolkits) [33] and displayed them with circus software of TBtools software.

Dual Synteny Plotter software was adopted to generate the syntenic relationships between XsLOX and LOX genes from Arabidopsis thaliana, Populus trichocarpa, and Oryza sativa; Ka and Ks substitutions, as well as the Ka/Ks ratio, were respectively determined using KaKs Calculator software 2.0 [34].

Analysis of cis-acting regulatory elements in XsLOX genes

The 2.0 kb upstream promoter sequences of each XsLOX genes were uploaded to the online site PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to identify the cis-acting regulatory elements.

Plant materials and stress treatments

Yellow horn seedlings grew in a greenhouse at 25°C for a month. Healthy seedlings were treated at 4°C, 150 mM NaCl, and 150 mM Na2CO3 (pH 9.5) as cold, salt, and saline-alkali stress, respectively. The leaves were collected at low temperatures at 0 h, 4 h, 12 h, and 24 h, and salt, and saline-alkali stress at 0 h, 4 h, and 24 h for transcriptome sequencing. All transcriptome data with accession numbers (salt, at SRX7830132—SRX7830143; alkali and low-temperature stress, at SRX8911738—SRX8911752) were available from the NCBI SRA database. We mapped these data to the X.sorbifolium reference genome [35] and evaluated XsLOX gene expression levels using Cufflinks software [36]. A heat map was generated using TBtools.

Four-weeks-seedlings were treated with 100 mM NaCl, 100 μM ABA, 200 μM Salicylic Acid (SA), 10% Polyethylene Glycol 6000 (PEG6000), 100 μM Gibberellin A3 (GA3), and 4°C as cold stress respectively. Twenty-four hours after exposure to the abiotic and hormonally medicated treatments, leaves were collected. Various tissues were obtained from the experiment plot to procure specimens at the flowering stage from triennial plants. Before RNA extraction, the collected samples were frozen directly in liquid nitrogen and stored at -80°C.

qRT-PCR was performed with Fast SYBR Green PCR kit on an ABI 7,500 apparatus (Applied Biosystems, Carlsbad, CA, USA), and PCR primer pairs for candidate genes were designed by using Primer3 (S1 Table). UBC2 (EVM0006862) was used as an internal control. Finally, the comparative 2−ΔΔCt method computed the relative expression, and each sample was replicated three times.

Results

For identification and classification of XsLOX

15 XsLOX gene members were identified from yellow horn genome through HMM-scan software and BLAST search at the genome-wide level. We named these LOX genes XsLOX1 to XsLOX15 according to their order on the chromosome positions. The fundamental properties of XsLOX genes were then analyzed and represented in S2 Table, including gene ID, gene name, position on the chromosome, length of coding sequence (CDS), length of the corresponding protein, molecular weight (MW), theoretical isoelectric point (pI), and the predicted subcellular localization. The related proteins of the XsLOX genes ranged in length from 831 (XsLOX1) to 927 (XsLOX11) amino acids, with their respective molecular weights and isoelectric points varying from 94.82 kDa to 104.52 kDa and from 5.33 (XsLOX14) kDa to 7.7 (XsLOX11) kDa, respectively. Most XsLOX genes were predicted to target the cytoplasm and chloroplast, while only one gene was localized within the nucleus.

Phylogenetic analysis of XsLOX

A neighbor-joining phylogenetic tree was constructed with 15 LOX sequences (from X.sorbifolium), 6 LOX sequences (from A. thaliana), 11 LOX sequences (from O. sativa), and 18 LOX sequences (from P. trichocarpa). Utilizing this tree, researchers were able to trace the evolutionary history of these genes (Fig 1). It showed that XsLOX genes were classified into three categories: type I 13-LOX, type II 13-LOX, and 9-LOX. Five and nine XsLOXs were assigned to the 9-LOX subfamily and type II 13-LOX subfamily, respectively. The type I 13-LOX subfamily was seen to be the minor branch occupied by a single gene.

Fig 1. Phylogenetic tree of LOX genes with other plants.

Fig 1

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The phylogenetic tree in the figure also distinguished the LOX genes of the various plants with distinct colors (9-LOX: purple, type I 13-LOX: orange, and type II 13-LOX: green). AtLOX: Arabidopsis thaliana. PtLOX: Populus trichocarpa. OsLOX: Oryza sativa.

Analysis of gene structure and conserved motif of XsLOX

We observed that the number of exons and introns varied among XsLOX genes and that conserved domains were broadly distributed (Fig 2). It was apparent that all XsLOX genes featured exons, nine being the most widespread within the family. XsLOX9 was the most abundant in exonic components compared to the other genes. Using MEME, we also revealed that the subfamily included ten conserved motifs in the LOX proteins. All ten motifs were present in all XsLOX proteins, and they had the same organization, indicating a considerable level of sequence similarity and conservation within XsLOX. Motif 1, consisting of HIS-(X) 4 -HIS-(X) 4 -HIS-(X) 17 -HIS-(X) 8 -HIS, is purported to be an integral pattern influencing the activity and stability of the lipoxygenase (LOX) enzyme [5, 37] (S3 Table).

Fig 2. Phylogenetic relationships, conserved domain regions, gene structure, and motif patterns of LOX genes in yellow horn.

Fig 2

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Clustal W and MEGA 7 were employed to realign the complete amino acid sequence and formulate the phylogenetic tree. The untranslated region (UTR), exons, and introns were depicted as green boxes, yellow boxes, and grey lines, respectively.

Chromosome distribution and duplication of XsLOX

The map chart software was employed to specify the genes’ locations on chromatids. Subsequently, the XsLOX genes were categorically renamed according to their chromosomal distribution. Data analysis suggested substantial divergence in the figure of XsLOX genes across chromosomes (Fig 3). Chromosome number 6 contained the most XsLOX genes, whereas chromosomes 1, 8, and 9 each harbored only one gene. Interestingly, the research did not demonstrate a conspicuous association between the length of chromosomes and the proliferation of XsLOX genes. The aggregate elucidation from the investigation indicated an asymmetric diffusion of XsLOX genes on the chromosomes.

Fig 3. Further illustrates the alignment of XsLOX genes in 5 chromosomes, expressing the gene arrangements in relative proportions.

Fig 3

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Each line on the chromosome represents the fragment of a gene. The correlation between the two gene headers indicates that they are matched duplicates.

Seven XsLOX genes were observed in the MCScanX package to have experienced successive duplication events across chromosomes, with three pairs constituting the 13-LOX II branch and one belonging to the 9-LOX branch in yellow horn (Fig 3). This duplication is correlated with an increase in the XsLOX family, and Ka/Ks ratios were calculated to explore the effects of selection on this gene family following replication (S4 Table). These figures suggest that the genes within the LOX genes are evolving under negative selection pressure, as evidenced by the Ka/Ks ratios that range from 0.24 to 0.39.

Every chromosome is denoted with a numerical label at the right. A syntenic map of three species (Fig 4) can be utilized to investigate the evolutionary relationship of the XsLOX genes. Collinearity analysis revealed that the four XsLOX genes were homologous with A. thaliana genes, two XsLOX genes with rice genes, and seven XsLOX genes with P. trichocarpa genes on the chromosome.

Fig 4. Homolinear LOX gene analysis between X.sorbifolium, rice, A. thaliana, and P. trichocarpa.

Fig 4

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A homology-based LOX gene analysis was performed among X.sorbifolium, rice, A. thaliana, and P. trichocarpa. The gray lines in the background indicated collinear blocks between X.sorbifolium, rice, A. thaliana, and P. trichocarpa, while the red line signified the collinear LOX gene pairs.

Analysis of cis-acting regulatory elements (CREs) of XsLOX

Cis-regulatory elements comprise non-coding DNA sequences, which can influence regulatory networks, and are bound by transcription factors to regulate gene expression. CREs have an integral role in regulating gene expression due to embedding in non-coding DNA regions, which are predominantly bound by transcription factors to modulate the transcription of adjacent genes. To uncover the cis-acting elements of XsLOX genes, the PlantCare program was employed to detect the 2000-bp genomic DNA upstream sequence of the start codon. The analysis showed that the cis-acting elements in the promoter regions were mainly associated with MeJA, ABA, IAA, GA, and SA, indicating that XsLOX genes may be involved in plant hormone signaling pathways (Fig 5).

Fig 5. Phylogenetic tree and distribution of cis-acting elements in the XsLOX gene promoter regions.

Fig 5

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Different colors in the boxes represent the cis-acting elements.

Moreover, the CREs of genes are linked to various conditions such as stress tolerance, healing, defense, hypothermia, anaerobic conditions, and hypoxia. Our examination of cis-regulatory elements uncovered discrepancies in the number of CREs among the five gene families: 119 primarily had abscisic acid response elements, 101 possessed MeJA response elements, 93 contained gibberellin response elements, 37 had low-temperature responsive elements, 145 had light-responsive features. Interestingly, soloist genes had fewer CREs when compared to other genes that were associated with hormone pathways and stress responses. Analysis of cis-regulatory factors revealed differences in the number of CREs among the five gene families. The outcome implies that XsLOX is essential for the growth and assimilation of plants in diverse environmental conditions.

For identification and classification of XsLOX

Transcriptome analysis of LOX

To comprehend the expression pattern of XsLOX genes, we analyzed RNA-seq data under salt, alkali, cold, and drought stress. The heat map was constructed with TBtools software.

The expression levels of each XsLOX gene were evaluated using RNA-seq data (Fig 6). When exposed to low-temperature stress, two XsLOX genes were upregulated, and five were downregulated at both 4 hours and 24 hours. Similarly, six XsLOX experienced decreased expressions, and four experienced increased expressions after 12 hours. By contrast, two and four genes were downregulated and upregulated when exposed to salt stress for 4 and 24 hours. Upon alkali stress for 4 hours, three XsLOX genes decreased, and three increased in expression. In addition, during the 24-hour stress-induced period, eight XsLOX remained downregulated, with none upregulated. Finally, one XsLOX gene was downregulated, and five upregulated after the application of drought. Among them, the three XsLOX genes (XsLOX5/ 7/ 9) co-expressed and downregulated in response to all treatments. These findings demonstrate that XsLOXs are pivotal actors in abiotic stress-related processes.

Fig 6. The differential expression levels of the XsLOX genes in response to low temperature treatment (LT), salt treatment (ST), and alkali treatment (AT).

Fig 6

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The seedlings were treated with NaCl (150 mM) and Na2CO3 (150 mM) for 4 h and 24 h, low temperature (4°C) for 4 h, 12 h, and 24h. Red squares indicate increased abundance and blue squares indicate decreased quantity.

qRT-PCR analysis of LOX

To further understand the expression pattern of the XsLOX gene in yellow horn, expression profiles of the XsLOX gene in seven different tissues were examined from qRT-PCR data (Fig 7). In standardizing relevant expression data, leaf expression is used as a reference. XsLOX1, XsLOX3, and XsLOX7 were highly expressed in roots, stems, axillary buds, and buds, while relatively few existed in other tissues. In particular, XsLOX7 was the highest expression level in flowers and stems, indicating that the XsLOX gene mainly plays a role in these tissues. It is worth noting that XsLOX5, XsLOX9, XsLOX10, XsLOX12, and XsLOX15 showed opposite expression patterns, and only XsLOX15 had slightly higher expression levels in axillary buds. The results of qRT-PCR showed that XsLOX had a significant tissue-specific expression, indicating the different roles in plant development.

Fig 7. Changes in expression analysis of eight randomly chosen XsLOX genes in seven tissues of mature plants by qRT-PCR.

Fig 7

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All qRT-PCR data were normalized against that of the housekeeping gene. X-axis shows different tissues, and Y-axis shows the relative expression level. Error bars represent the mean ±SE of three biological replicates, and asterisks represent a significant difference from Duncan’s method (*p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001).

qRT-PCR was used to verify the expression pattern of XsLOX under abiotic stress and hormone. After drought treatment, XsLOX1 was up-regulated, XsLOX5 and XsLOX9 were down-regulated, and other genes were not significantly expressed (Fig 8). The expression of XsLOX5/9/10/12 (type II 13-LOX) was up-regulated considerably under dark and low-temperature treatment. XsLOX7 is highly expressed under dark processing. After salt treatment, only XsLOX3 was upregulated. The expression levels of XsLOX1 and XsLOX3 have been up-regulated after SA and GA treatment, but no significant induction of any XsLOX genes was observed after ABA treatment. XsLOX genes play a potential role in combating abiotic stress and responding to exogenous plant hormones.

Fig 8. Changes in expression analysis of eight randomly chosen XsLOX genes in response to 10% PEG 6000, 100 mM NaCl, 4°C low temperature, 100 μM ABA, 200 μM SA, and 100 μM GA stress for 24 h and dark for 48 h.

Fig 8

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X-axis shows the different stress, and Y-axis shows the relative expression level. Error bars represent the mean ±SE of three biological replicates, and asterisks represent a significant difference from Duncan’s method (*p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001).

Discussion

Lipoxygenases have been the subject of intensive study in various organisms, playing a critical role in growth and development, signaling molecule generation, stress response, and plant defense [3, 17, 38, 39]. Through the utilization of HMM and BlastP combining the annotation data from yellow horn, fifteen potential XsLOX genes have been uncovered, surpassing those found in Arabidopsis thaliana [40] and tea plant [41], foxtail millet [16], tartary buck wheat [42], Passiflora Edulis [27], tomato [12], maize [43], but still be less than those discovered in Salvia miltiorrhiza bung [44], banana [45], apple [9], cucumber [39], cotton [15] and Populus [46]. An evolutionary analysis revealed that the four XsLOX proteins comprising XsLOX2/3/4/13/14 assemble into the 9-LOX subfamily; XsLOX1 falls under the 13-LOX I subfamily, with XsLOX5/6/7/8/9/10/11/12/15 conforming to the 13-LOX II subfamily [5, 9]. The familial evolution of genes has been primarily ascribed to gene duplication through consecutive repetition events. Evolutionarily, variance in genetic phylogenetic trees may suggest conflict in active roles, while genes clustered on the same branch may have a similar function.

XsLOX3/4 and XsLOX5/6/7/8/9 are disposed of in tandem on chromosomes 3 and 7, respectively. The evolution of genes has been predominantly correlated to the duplication of the gene through consecutive replication events. This system may produce new genes and sub-activities, devising complementary non-redundant uses with the ancestral genes. Tandem echo can take place for multiple motives. It is assumed that unequal chromosomal crossings are the main catalysts of duplication events and the generation of genetic disparities among the species.

It is postulated that the presence of tandem repeated genes facilitates the organism’s adaptability to changing conditions, playing an essential part in evolutionary parsimony. Tandem repeats are believed to contribute to the prokaryote organism’s resilience and invulnerability, a claim corroborated by the fact that the expression of LOX genes is involved [47]. The Ka/Ks ratio was observed to be inferior to one, thus providing pointers for research related to the genes of yellow horn immunity [48].

Subcellular localization prediction analysis of XsLOX indicated that type LOX II proteins possess an N-terminal plastid transporter and are localized in the chloroplast, consistent with the related family clustering outcomes. In contrast, type LOX I proteins are mainly found in the cytoplasm, lacking the transporter [49].

The gene structure and lipoxygenase domain were highly conserved in XsLOX genes. To gain insight into how the gene is regulated under various environmental conditions, we analyzed promoter cis-acting elements of each XsLOX gene family. Our findings indicate cis-elements were classified into MeJA response elements, gibberellin response elements, low-temperature response elements, drought-inducibility elements, circadian control, light-responsive elements, and hormonal pathways. These outcomes align with those observed in other species [13], with the most light-sensitive features.

LOX plays an important role in the growth and development of yellow horn. The expression levels of some genes detected by transcriptome sequencing did not match the qRT-PCR analysis. By qRT-PCR analysis, it was found that the expression levels of one down-regulated gene XsLOX12 under low-temperature stress were different from the RNA-Seq data. LOX can catalyze both enzymatic and non-enzymatic pathways to synthesize oxidized lipids. The expression of this enzyme and its gene underlies the synthesis of these lipids and stress responses. It is speculated that this gene’s expression may result from its involvement in specific metabolic pathways correlated with the synthesis of certain molecules. The AtLOX1 gene has been demonstrated to be critical in maintaining defensive responses in Arabidopsis leaves [22]. AtLOX5 has been identified as highly expressed in the roots and appears to be involved in lateral root morphogenesis [14]. Studies have indicated that enhanced 9-LOX catalysis of 9S-HPOT synthesis, with its derivatives, has been linked to the dynamics of lateral root formation and could thus reduce primary root growth [22, 50]. Tomato TomLoxB and TomLoxC positively regulated ethylene content during fruit ripening [51, 52]. The application of jasmonic acid promoted the expression of ZmLOX6 in maize [53]. JA and SA induce the expression of Gllox1 and Gllox2 in Gracilariopsis lemaneiformis [54]. In our study, SA induces the expression of XsLOX1 and XsLOX3. In injured potatoes and rice, ABA can stimulate LOX activity [55, 56]. The GhLOX gene plays an important role in the development of the nutrient tissue of cotton [15]. Expressions of XsLOX1, XsLOX3, and XsLOX7 were significantly elevated in axillary buds, stems, and roots, suggesting that these genes may have important functions in these tissues.

Conclusions

In this study, 15 XsLOX genes were identified, exhibiting highly conserved gene structures and being divided into two subgroups and unevenly distributed across a single chromosome. Analysis of their differential and specific expression under heat, cold, drought, and salt stress and subsequent qPCR verification revealed they play a significant role in responding to abiotic stresses. Moreover, XsLOX genes were found to be particularly active in tissue specificity, which may be correlated to their structural similarity among family members and differences in the cis-acting elements. Consequently, our findings provide new insights into the crucial activities of the XsLOX gene across evolution, development, and abiotic stress tolerance.

Supporting information

S1 Table. q-PCR primers.

(PDF)

Click here for additional data file. (68.4KB, pdf)
S2 Table. List of XsLOX genes identified.

(PDF)

Click here for additional data file. (98.2KB, pdf)
S3 Table. Motif 1 contains the conserved sequence His-(X) 4 -HIS-(X) 4 -HIS-(X) 17 -HIS-(X) 8 -HIS.

(PDF)

Click here for additional data file. (95.6KB, pdf)
S4 Table. Estimation of the Ka/Ks ratio of homologous gene pairs in yellow horn.

(PDF)

Click here for additional data file. (81.5KB, pdf)

Data Availability

The raw RNA-seq data are available in the National Center for Biotechnology Information (NCBI) under SRA accession number: PRJNA759108.

Funding Statement

The author(s) received no specific funding for this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. q-PCR primers.

(PDF)

Click here for additional data file. (68.4KB, pdf)
S2 Table. List of XsLOX genes identified.

(PDF)

Click here for additional data file. (98.2KB, pdf)
S3 Table. Motif 1 contains the conserved sequence His-(X) 4 -HIS-(X) 4 -HIS-(X) 17 -HIS-(X) 8 -HIS.

(PDF)

Click here for additional data file. (95.6KB, pdf)
S4 Table. Estimation of the Ka/Ks ratio of homologous gene pairs in yellow horn.

(PDF)

Click here for additional data file. (81.5KB, pdf)

Data Availability Statement

The raw RNA-seq data are available in the National Center for Biotechnology Information (NCBI) under SRA accession number: PRJNA759108.

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