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Gibberellin Regulates LBD38-1 Responses to Xanthomonas arboricola pv. juglandis Infection in Walnut Bacterial Blight Pathogenesis

BMC Genomics volume 26, Article number: 370 (2025) Cite this article

Abstract

Background

Plant responses to biotic and abiotic stresses are complex processes. Previous studies have shown that the LBD gene family plays important roles in plant growth and development as well as in plant defense against biotic and abiotic stresses. The expression of LBD genes was investigated in walnuts under biotic and abiotic stresses, revealing that LBD38-1 may be a key gene in the plant stress response. This study provides new insights into the roles of LBD genes in plant responses to biotic stress.

Results

Forty-nine members of the JrLBD gene family were identified in the walnut genome and classified into six subfamilies. Comparative homology analysis through phylogenetic trees revealed that the presence of Group I-a and Group VI plays an important role in resistance to stressors. The expression of walnut LBD genes under cold-temperature, high-temperature, mechanical damage, and biotic stresses was analyzed via transcriptome sequencing, and the expression of JrLBD38-1 in the Group VI subfamily was particularly prominent. According to transcriptome profile analysis, JrLBD38-1 is highly expressed in different tissues of walnuts, suggesting that it plays a regulatory role in the growth and development of different tissues. The function of the Gibberellin (GA) response element in the JrLBD38-1 promoter was further analyzed and verified. These findings confirmed that GA regulated JrLBD38-1 expression changes during Xanthomonas arboricola pv. juglandis infestation of walnut leaves.

Conclusion

Forty-nine walnut JrLBDs were identified and classified into six subfamilies. JrLBD38-1 has GA-inducible expression, is regulated by GA under pathogenic bacterial stress, and is involved in the response to biotic stress. This function of JrLBD38-1 provides new insights into walnut disease resistance mechanisms.

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Background

Walnuts (Juglans regia L.), also known as English and Persian walnuts, are deciduous trees of the family Juglandaceae. Walnuts are rich in protein, fiber, polyphenols, alpha-linolenic acid, and many other nutrients that are important for human health [1, 2]. The cultivation history of walnuts in China dates back more than 2000 years [3]. In recent years, the frequent climate anomalies, drought, heat, waterlogging, and cold damage has increased the attention of researchers on the response mechanisms of walnuts to biotic and abiotic stresses [4,5,6]. On the other hand, China's promotion of varietalization of walnuts and large-scale planting of precocious cultivars have led to the rapid expansion of precocious cultivars with poor resistance, resulting in the occurrence and gradual aggravation of diseases such as blight and anthracnose, which have severely affected the quality and yield of walnuts [7, 8].Walnut blight, caused by Xanthomonas arboricola pv. juglandis (Xaj.), is one of the most common infectious diseases of walnuts, causing severe economic losses. Therefore, considerable effort should be invested in understanding the innate resistance mechanisms in plants.

Lateral organ boundary (LBD) genes are a class of transcription factors that regulate the development and morphogenesis of plant lateral tissues and organs, and as the LBD gene family is present only in plant genomes, it may be involved in plant-specific regulation [9]. The LOB domain contains three motifs: the CX2CX6CX3C motif consists of four fully conserved cysteine residues separated by several conserved amino acid residues; the Gly-Ala-Ser motif consists of glycine, alanine, and serine; and the leucine-like zipper-like motif (LX6LX3LX6L) [10, 11]. Members of the LBD gene family are divided into two subfamilies depending on the completeness of the LOB domain that they contain: Class I genes contain the complete LOB domain, and Class II genes lack the Gly-Ala-Ser motif [11].

The LBD family of genes is widely distributed in many plants, such as Arabidopsis [12], Zea mays [13], Triticum aestivum [14, 15], Solanum tuberosum [16], and Vitis vinifera [17]. In recent years, the LBD gene family has been reported to be closely associated with plant growth and developmental regulatory processes, such as secondary growth, fruit growth and development, the environmental stress signal response, and the pathogen response [18,19,20,21,22,23]. ARF7/ARF19-LBD16/LBD18, via AUX1/LAX3 auxin influx carriers, plays a pivotal role in lateral root formation and is involved in AR formation in Arabidopsis [24]. In Vitis vinifera [17] and Solanum lycopersicum [25], LBD transcription factors are involved in fruit development, berry ripening, and hormone response [26]. Specifically, LBD plays a regulatory role in the response to biotic and abiotic stresses [27]. SlLBD40 is involved in JA signaling and is a negative regulator of drought resistance in tomato [28], and SlLBD40 knockout enhances drought resistance in tomato. On the other hand, LBD genes are involved in regulatory processes in response to biotic stress. LBD20 may function in jasmonate (JA) signaling, and disruption of the LBD20 gene leads to increased resistance to the root-infecting vascular wilt pathogen [29]. PthA4, the effector gene of the Xanthomonas citri subspecies, enters the citrus genome, where it specifically recognizes and binds to the CsLBD1 gene promoter, regulates the expression of CsLBD1 in the host, and increases susceptibility to citrus bacterial canker disease [30]. Genes in the LBD family are also affected by GAs during the regulation of plant growth, development, and resistance to stress. ZmLBD5 is involved in the regulation of maize (Zea mays) by affecting the synthesis of GAs and abscisic acid (ABA) through the regulation of maize growth and the drought response by affecting the synthesis of GAs and ABA [31]. In transgenic rice plants heterologously overexpressing PheLBD12, GA3 bioactivity was reduced, and the internodes were shortened [32].

The central role of the GA class of growth hormones in the response to abiotic and biotic stress is becoming increasingly evident [33, 34]. Genes and regulatory proteins involved in GA synthesis and signaling pathways are closely related to plant stress tolerance [33, 35]. Plants can adapt to unfavorable external conditions by regulating the expression of GA-related metabolic genes, such as GA20oxs, GA3oxs, and GA2oxs, and GA signaling-related proteins, such as DELLAs [36]. In addition, plants can improve salt tolerance by reducing active GAs [37]. The overexpression of GA2oxs in rice enhances salinity tolerance, which is lost when GA3 is applied externally [38]. Similarly, GAs play an important role in the plant response to biotic stress. Exogenous GA3 treatment of Nicotiana benthamiana plants increased their susceptibility to the infectious pathogens Ralstonia solanacearum and Rhizoctonia solani [39]. In particular, the exogenous application of GA3 and an inhibitor of GA synthesis (uniconazole) can increase disease susceptibility and resistance to bacterial blight, respectively [40].

In this study, we performed a systematic analysis of LBD family genes at the genomic level, including chromosomal localization, phylogenetic tree construction, gene structure analysis, conserved motif detection, and promoter element analysis, via published walnut genomic data. Changes in the expression of walnut LBD family genes in response to cold-temperature, high-temperature, mechanical damage, and biotic stresses were investigated, and the stress-sensitive response gene JrLBD38-1 was screened by combining the expression differences of LBD genes in different tissues. Furthermore, expression analysis of JrLBD38-1 and functional validation of promoter cis-acting elements confirmed that GA participates in the response to biotic stress by regulating the LBD. This study clarifies the relationship between GA and LBD and provides new insights into the role of GA and LBD genes in plant responses to biotic and abiotic stresses.

Results

Identification and analysis of LBD genes in the walnut genome

In this study, 49 JrLBD genes were identified from the walnut 2.0 genome, and these 49 family members were named according to their covariance with Arabidopsis (Supplementary file 1). Among all the JrLBD genes, the longest gene length was 27,203 bp (JrLBD21), the shortest length was 390 bp (JrLBD22-4), the length of the cds sequences formed after coding was the longest at 1266 bp (JrLBD36-4) and the shortest at 390 bp (JrLBD22-4), the translated length of the protein was between 129 and 421 aa, and the theoretical molecular weight was between 14.84 kDa and 46.80 kDa. There were 29 proteins with an isoelectric point (pI) < 7, of which the smallest was 4.97 (JrLBD33), and 20 proteins had an isoelectric point (pI) > 7, of which the largest was 9.88 (JrLBD22-4). Subcellular localization prediction revealed that all JrLBD family genes were localized in the nucleus, indicating that LBD transcription factors can function in the nucleus.

Phylogenetic trees are widely used to show evolutionary relationships among genetic families. To study the evolutionary relationships between LBD family genes in walnuts and Arabidopsis thaliana, a phylogenetic tree was constructed via the neighbor‒joining (NJ) method in the MEGA 11 program with the full-length proteins of 49 genes in walnut and 42 genes in Arabidopsis thaliana. According to the phylogenetic tree, the 49 members of the walnut LBD family were divided into six groups, Groups I-VI, of which Group I was divided into Group I-a and Group I-b. Among them, subfamily II was the largest, with significant gene duplication and extension. Group II was further divided into groups II-a, II-b, II-c, and II-d (Fig. 1).

Fig. 1
figure 1

Phylogenetic analysis of LBD proteins in Arabidopsis thaliana and Juglans regia. Areas of different colors represent different subfamilies. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches

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Chromosomal analysis and collinearity analysis of walnut JrLBD members

Chromosomal localization analysis revealed that the JrLBD family genes presented a random and uneven distribution on the remaining 15 chromosomes, except of chromosome 16 (Supplementary file 2). The chromosome numbers with the highest to lowest numbers of genes were, in descending order, chromosomes 7, 10, 12, 1, 14, 2, 11, 8, 5, 3, 15, 13, 9, 6, and 4, with a total of 10 genes on chromosome 7, which had the highest number of genes in the distribution, and only one gene on chromosomes 15, 13, 9, 6, and 4. Among the 49 JrLBD family members identified, more than half were present in gene clusters.

The within-genome duplication events of the JrLBD genes were analyzed via MCScan. The results indicated that six genes located on chromosomes 1, 7, and 10 were involved in tandem duplication events, resulting in the formation of three gene pairs. We also performed collinearity analysis of the LBDs to explore the evolutionary relationships of the JrLBDs. The results revealed that 11 syntenic JrLBD gene pairs derived from whole-genome duplication or segmental duplication were distributed on 13 of the 16 chromosomes (Fig. 2).

Fig. 2
figure 2

Distribution of LBD genes derived from segment replication in the walnut genome. The red lines represent the pairing of the JrLBD gene, whereas the gray lines represent the pairing of all the walnut genes. The outer ring represents the position of JrLBDs on each chromosome. Boxes of different colors represent the 16 chromosomes of walnuts

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Conserved motif and gene structure analyses of the walnut LBD gene family

To determine the genetic structure and evolutionary trajectory of the JrLBD gene in walnut, the exon‒intron compositions of 49 JrLBDs were studied. The gene with the greatest number of introns was JrLBD36-4, with five, and there were no introns in JrLBD2-1 or JrLBD22-4. In addition, the genes forming the subgroups presented similar gene structures (Fig. 3a, b).

Fig. 3
figure 3

Conserved motif analysis and gene structure analysis of walnut LBD members. a Gene structure analysis. b Conservative motif analysis. The number represents the branch series

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The conserved motifs of JrLBD proteins were predicted. Ten conserved motifs, named 1–10, were identified in this study (Supplementary file 3). All the proteins encoded by the JrLBD family genes contained the predicted motif 1 and motif 3, and the number and type of conserved motifs in the protein sequences were relatively consistent across proteins. The class I members had motif 1 (CX2CX6CX3C), motif 3 (Gly-Ala-Ser), and motif 4 (LX6LX3LX6L), whereas the class II members lacked motif 4 (LX6LX3LX6L), a result that is consistent with the findings of previous studies.

Expression patterns of JrLBD genes under biotic and abiotic stress

The transcriptome data of walnut were used to analyze the gene transcription and expression patterns of the JrLBD family in the 'Qingxiang' cultivar under cold-temperature stress for 48 h (Fig. 4a). The results revealed that the transcription levels of JrLBD38-1 and JrLBD38-4 significantly increased after 0.5 °C low-temperature stress treatment for 0 h and 48 h, and the expression level of JrLBD38-1 significantly increased, making it the most highly expressed gene of the LBD transcription factor family.

Fig. 4
figure 4

Expression patterns of JrLBD genes under biotic and abiotic stresses (a). Changes in JrLBD expression during cold stress. b Changes in JrLBD expression during heat stress. c Changes in JrLBD expression during mechanical injury stress. d Changes in JrLBD expression during biotic stress

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The transcriptome data of walnut were used to analyze the gene transcription and expression patterns of the JrLBD family in the 'Qingxiang' cultivar under heat stress for 6 h (Fig. 4b). The results revealed that the expression of JrLBD38-1 changed the most after 0, 2, and 6 h of high-temperature stress in the potted walnut seedlings. The expression of JrLBD38-1 significantly decreased after 2 h of high-temperature stress.

The transcriptome data of the walnuts were used to analyze the gene transcription of the JrLBD family under mechanical damage stress (Fig. 4c). The results revealed that the expression levels of JrLBD38-1, JrLBD41-1, JrLBD41-2, and JrLBD41-3 increased to different degrees after 12 h of injury stress, with the expression level of JrLBD38-1 being the highest. The expression levels of JrLBD38-1, JrLBD41-1, and JrLBD41-3 were lower than those at 12 h after 24 h of injury but were still higher than those at 0 h, and the expression levels of JrLBD41-2 increased. The expression of JrLBD38-1 was the highest 24 h after injury.

The transcriptome data of walnuts were used to analyze the transcription patterns of JrLBD family genes under biological stress (Fig. 4d). The transcript levels of JrLBD38-1 tended to decrease but then increase, whereas those of JrLBD1-3 tended to increase but then decreased.

Expression analysis of JrLBDs in different walnut tissues

The transcriptome data of seven different tissues of 'Qingxiang' plants (pistils, catkins, roots, mature leaves, young leaves, young stems, and buds) revealed differences in JrLBD expression (Fig. 5). JrLBD38-1 presented the highest expression in roots, catkins, and young leaves. JrLBD38-4 was highly expressed in mature leaves, and JrLBD41-1 was highly expressed in roots. Multiple genes belonging to the Group VI subfamily presented high expression levels in different tissues (Supplementary file 4). The expression of genes from different subfamilies also significantly differed across different tissues. These findings suggest that JrLBDs play important roles in tissue formation and differentiation. Therefore, the present study was further conducted on JrLBD38-1 on the basis of its differential expression in different tissues and under different stresses.

Fig. 5
figure 5

Transcriptional expression of JrLBD gene family members in different walnut tissues (young stems, buds, young leaves, mature leaves, pistils, catkins, and roots)

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Changes in the GA content and expression of JrLBD38-1 during bacterial stress treatment of leaves

Since the expression of JrLBD38-1 changed significantly under different stress treatments, to further determine its bacterial stress response, RNA was extracted from the leaves at different times after inoculation (Fig. 6a), and cDNA was obtained via reverse transcription. qRT‒PCR was performed on the JrLBD38-1 gene, and the results revealed that the expression of JrLBD38-1 tended to decrease from 0–24 h, after which the expression increased over time, which was generally consistent with the transcriptome sequencing results. (Fig. 6b).

Fig. 6
figure 6

a Leaf changes under different inoculation treatment times. b Relative expression of JrLBD38−1. The bars represent the means ± SDs (n = 3). Significant differences between treatments were determined by Student's t test: * < 0.05. ** P < 0.01. *** P < 0.001, Student's t-test. The same as below. c Analysis of the promoter cis-acting element analysis of JrLBD38-1. Different colors represent different components. The red star marks the original response element of gibberellin. d Variation in the GA content

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Hormone levels play an important role in plant responses to pathogen stress. Generally, gene function is affected by the distribution of cis-acting elements in the promoter region. According to the predicted gene structure of the members of the JrLBD gene family, the upstream 2000 bp of the CDSs were extracted as the promoter sequence. Analysis of the promoter cis-acting elements of JrLBD38-1 revealed that among the hormone-related cis-acting elements, the number of GA-responsive elements was the greatest (Fig. 6c). Thus, the content of GAs in the inoculated leaves was determined, and the results revealed that the GA content in the inoculated leaves gradually increased from 0 to 48 h with increasing treatment time and then decreased to the initial level after 72 h of treatment. Quantitative analysis of JrLBD38-1 revealed that both JrLBD38-1 expression and the GA content peaked at 48 h after inoculation (Fig. 6d). Therefore, we suggest that the expression of JrLBD38-1 is regulated by GA and that the GA response element in the JrLBD38-1 promoter may be responsible for this regulation.

Analysis of the locus of GA affecting LBD expression

To further explore the role of GA response elements in the JrLBD38-1 promoter, a recombinant vector of the JrLBD38-1 promoter-pGREEN II 0800-LUC was constructed, and luciferase was instantly transformed into tobacco (Fig. 7a). The fluorescence results revealed that the fluorescence intensity of the Q0 segment of the JrLBD38-1 promoter significantly increased after GA treatment. The fluorescence intensity of the promoter Q1 segment also significantly increased after GA treatment. The fluorescence intensity of the Q2 segment of the JrLBD38-1 promoter did not change significantly after GA treatment (Fig. 7b and c). After the GA element in the JrLBD38-1 promoter fragment was removed (Fig. 7d), the fluorescence results revealed that the fluorescence intensity of the Q1* and Q2* segments of the JrLBD38-1 promoter did not change significantly after GA treatment. The fluorescence self-activation of the Q2* segment of the JrLBD38-1 promoter was stronger than that of the Q2* segment of the JrLBD38-1 promoter (Fig. 7e and f). On the basis of the fluorescence invariant results of different segments, we determined that the JrLBD38-1 promoter was responsive to GA treatment and that the responsive segment was Q1. In summary, JrLBD38-1 exhibits GA-induced expression, is regulated by GA under pathogen stress, and is involved in the response to biotic stress.

Fig. 7
figure 7

Analysis of the cis-regulatory elements of the promoter. a Schematic representation of the reporter plasmids used in the dual LUC assay (retaining the gibberellin-responsive originals Q0, Q1 and Q2). b Relative activity levels of LUC (Q0, Q1 and Q2). Each bar indicates the mean ± SD from three biological replicates (∗ ∗ ∗ P < 0.001). c Analysis of the Dual Luciferase Assay System (Q0, Q1, Q2, CK: sterile water application treatment as the control group; GA: 100 mg/L GA solution-coated treatment as the experimental group.) d Schematic representation of the reporter plasmids used in the dual LUC assay (removing the gibberellin-responsive originals Q1*, Q2*). e Relative activity levels of LUC (Q1*, Q2*). Each bar indicates the mean ± SD from three biological replicates (∗ ∗ ∗ P < 0.001). f Analysis of the Dual Luciferase Assay System (Q1*, Q2*, CK: sterile water application treatment as the control group; GA: 100 mg/L GA solution-coated treatment as the experimental group.) Note: Q0 is the promoter fragment −1,150 bp to −125 bp, Q1 is the promoter fragment −1150 bp to −584 bp, and Q2 is the promoter fragment −604 bp to −125 bp

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Discussion

As plant-specific transcription factors, LBD family members have been extensively studied in plants. To date, the LBD gene family has been identified and characterized in many plants, including 56 LBDs in Solanum lycopersicum [41], 55 in Phyllostachys edulis [42], 67 in Malus domestica [43] and 41 in Rosa rugosa [44]. In this study, 49 LBD family members were identified and analyzed on the basis of their protein properties. In addition, a similar genetic structure was observed for each small branch, with high subfamily conservation. Fragmentary and tandem replication of duplicated genes are the two main causes of gene family expansion in plants [45, 46]. There are 24 pairs of LBD gene replication sequences in the Ginkgo genome, including 22 pairs with fragment replications and only two pairs with tandem replications [47]. In contrast, there were 11 groups of collinear genes among the members of the walnut LBD gene family, and analysis of the collinear regions revealed that most of the genes probably originated from whole-genome duplication or gene fragment duplication.

A study of wheat LBD family genes revealed that the expression of some genes was affected by temperature, and the expression of two genes, Ta-2B-LBD73 and Ta-4D-LBD52, was greater at 4 °C than at 23 °C [48]. This study also confirmed that low- and high-temperature stress are involved in regulating the expression of LBD family genes. However, cold-temperature and high-temperature treatments had opposite effects on JrLBD38-1 expression, with cold-temperature treatment increasing JrLBD38-1 expression and high-temperature treatment decreasing JrLBD38-1 expression, suggesting that JrLBD38-1 expression is strictly regulated by temperature and that JrLBD38-1 may function under low- or high-temperature stress. This may be related to the role of the low-temperature response element in the JrLBD38-1 promoter.

Further expression analysis and promoter function verification of the LBD38-1 gene revealed that more important promoter elements, including the GA response, drought response, and ABA response elements, exist in the promoter region of the gene. The presence of these important response elements suggests a role for the LBD family in the response to hormonal signaling. The promoter of the citrus stress resistance gene CcGASA4 contains a GA response element (GARE), and exogenous GA3 induces the expression of this gene in the leaves, suggesting that GA can respond to stress responses by regulating functional genes [49]. CsLBD38 and CsLBD39-2 expression is upregulated in tomato after methyl jasmonate (MeJA) treatment, SlLBD40 is involved in the JA signaling pathway, which enhances drought resistance in tomato, and the ABA signaling pathway has been shown to regulate AtLBD40 in response to salt stress [50]. Therefore, we hypothesized that JrLBD38-1 is regulated by hormones through a response element in the promoter region, which in turn responds to biotic stress.

GAs play signaling roles in plant responses to biotic and abiotic stresses [33]. Drought-induced GA deactivation in guard cells contributes to stomatal closure in the early stages of soil dehydration, whereas the inhibition of GA synthesis in leaves suppresses canopy growth and restricts area [33]. GA also plays a key role in root-to-shoot communication to induce stomatal closure under water deficit conditions [35]. Exogenous GA3 treatment increases the susceptibility of tobacco to the pathogens Ralstonia solanacearum and Rhizoctonia solani [39]. In this study, significant changes in the GA content occurred after Xaj. infestation of walnut leaves, which was synchronized with the changes in the relative expression of JrLBD38-1. We also found that the promoter of JrLBD38-1 contained GA-responsive elements and was regulated by GA. In turn, changes in the GA content are affected by pathogen stress, and the GA content increases at the beginning of pathogen infection. To date, studies on the hormonal responses of LBD family genes have revealed that the Zea mays LBD family genes LBD5 and LBD33 regulate GA synthesis [31]. Therefore, we suggest that the expression of JrLBD38−1 increases in response to pathogenic bacterial stress by regulating the GA signaling pathway at an early stage of pathogen infection. In addition, we are also concerned that ABA- and SA-related response elements are also included in the cis-acting elements of the JrLBD38-1 promoter, and whether ABA and SA regulate JrLBD38-1 to cope with pathogenic bacterial infestation has become the focus of future studies.

Conclusion

Forty-nine walnut JrLBDs were identified and classified into six subfamilies. Xaj. infestation resulted in changes in the GA content of the walnut leaves. JrLBD38-1 has GA-inducible expression, is regulated by GA under pathogenic bacterial stress, and is involved in the response to biotic stress. The mechanism of the LBD family in response to disease resistance should be further elucidated. In conclusion, the discovery of JrLBD38-1 provides new ideas for the genetic improvement of disease resistance in walnuts.

Methods

Plant materials

The plant materials used in this study were 'Qingxiang' walnut (Juglans regia) and tobacco (Nicotiana benthamiana). The potted seedlings used for the low-temperature treatment were 'Qingxiang' two-year-old seedlings cultured in an incubation room at constant temperature and humidity (22 ± 2 °C, 70 ± 5%). Walnut explants were used for mechanical damage treatment (taken from the annual branches of 'Qingxiang' trees grown in the Experimental Field of Hebei Agricultural University, sterilized and cultured). Walnut tissues (young stems, mature stems, young leaves, mature leaves, pistils, catkins, roots and shoots) were collected from different seasons. For biotic stress, uniformly sized leaves were collected from potted seedlings of ' Qingxiang ' walnut (Supplementary file 5).

Stress treatments

To investigate the gene expression patterns of 'Qingxiang' walnut (Juglans regia) under various biotic and abiotic stresses across different tissues, we conducted stress treatments on potted walnut seedlings. On the basis of previous findings demonstrating significant physiological changes in walnut seedlings exposed to low temperature stress, we implemented a controlled low-temperature treatment (0.5 °C for 48 h) in a constant-temperature incubator. This treatment was applied to entire potted walnut seedlings to simulate cold stress conditions.

In parallel, considering the well-documented response of Arabidopsis thaliana to high-temperature stress [51], we designed a heat stress experiment in which walnut tissue cultures were used. Stem tip tissues of 'Qingxiang' walnut were cultured in DKW media for 28 days, followed by heat treatment at 42 °C for various durations (0 h, 2 h, and 6 h). This experimental design allowed us to examine the temporal effects of heat stress on walnut tissue cultures while maintaining controlled environmental conditions.

Walnut explants were cut and cultured in walnut agar media, and those subjected to mechanical damage treatments were sampled at 0, 12 and 24 h. The main causal agent of walnut black spot disease, Xanthomonas arboricola pv. juglandis, was isolated from walnut fruits in a diseased state, cultured and inoculated into walnut leaves (5 inoculations along the left and right sides of the main leaf veins by the needle-puncture method), and 1 cm2 of leaves around the diseased spots were collected at 0, 24, 72, and 120 h. All the plant materials were set up in triplicate, frozen in liquid nitrogen, and stored at −80 °C for testing.

Identification of the LBD gene family and bioinformatic analysis

The walnut genome used for genome-wide identification of the LBD transcription factor family in this study was derived from the NCBI database (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001411555.2/). The Arabidopsis LBD TF protein sequences (Supplementary file 6) were downloaded from the UniProt database (http://www.uniprot.org), and HMMER 3.1b software was used to construct a model with the Arabidopsis LBD TF protein sequences via the hmmbuild command to preliminarily screen for protein sequences containing LBD structural domains in walnuts (e-value < 1e-20). Moreover, via BLASTP, the walnut LBD TF sequences were screened using the Arabidopsis LBD TF protein sequences as seed sequences, the sequences obtained from the intersection of the two screenings were taken, and the structural domain information was viewed via the CDD-search tool and Pfam database (http://pfam.org/). After the sequences with redundancy and structural domain mutations were manually removed, the final walnut LBD transcription factor family was obtained.

The CDSs of the genes encoding the JrLBD family were submitted to GSDS 2.0 (https://gsds.gao-lab.org/) to analyze the gene structures. Two thousand bp upstream of the CDSs were used as the promoter region, and the CDSs were analyzed via Plant-CARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict their cis-acting element situation. TBtools software [52] was used to map the distribution of the cis-acting elements of the JrLBD38-1 promoter. The MEME website (https://meme-suite.org/meme/tools/meme) was used to predict the conserved motifs. The Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) online tool was used to predict the subcellular localization of family members. Phylogenetic analysis was performed via MEGA 11 (neighbor joining, bootstrap value of 1000), and covariance analysis within species of the JrLBD family was performed via MCScanX. The figures were processed via Adobe Illustrator.

Gene expression analysis

Total RNA was extracted from leaves treated with bacteria for different durations via the RNAprep Pure Plant Kit (Tiangen Biotech, China). cDNA was obtained via reverse transcription via the PrimeScript™ RT reagent Kit with gDNA Eraser Perfect Real Time (Tiangen Biotech, China). cDNA was used as a template, and JrActin [53] was used as an internal control. qRT‒PCR was performed on JrLBD38-1 via the specific primer (Supplementary file 7), and three independent experiments were performed to ensure reproducibility of the qRT‒PCR results (Supplementary file 8). The changes in the relative expression of genes under different treatments and treatment times were determined via the 2−ΔΔCt method.

Extraction and content determination of GA

To detect the content of GA in leaves at different times after inoculation, 0.5 g of plant samples at different times (0 h, 24 h, 48 h, and 72 h) were weighed and placed in a 10 mL centrifuge tube. After rapid freezing in liquid nitrogen, the samples were crushed via a fully automatic sample grinder. Sample powder (0.5 g) and 80% precooled (< 0 °C) methanol (4 mL) were added to 200 μL of the antioxidant BHT and mixed well at 4 °C, avoiding light extraction for 12 h, low-temperature ultrasonication without light for 1 h, and low-temperature centrifugation at 12,000 rpm/min at 4 °C for 15 min. The supernatant was transferred, and nitrogen was blown to the aqueous phase (approximately 0.8–1 mL; the aqueous phase was adjusted to pH 8 with 1 mol/L sodium dihydrogen phosphate (30–50 μL)) (excess was adjusted back with 1% acetic acid). The aqueous phase was adjusted to pH 8 with 1 molar of sodium dihydrogen phosphate (30–50 μL) (excess was adjusted back with 1% acetic acid). A total of 0.1 g of cross-linked polyvinylpyrrolidone (PVPP) was added to remove phenolic impurities. The mixture was ultrasonicated at low temperature in the dark for 0.5 h and then centrifuged at 12,000 rpm at 4 °C for 5 min at low temperature, after which the supernatant was transferred. The aqueous phase was adjusted with 1 mol/L citric acid to pH 3 (excess of which can be adjusted back with 1% sodium hydroxide) and added to 3 mL of the ethyl acetate extract. The mixture was then ultrasonicated for 10 min at a low temperature, protected from light, and allowed to stand for 2 h, after which the upper liquid nitrogen was transferred to the upper liquid nitrogen. The upper liquid nitrogen was transferred, the mixture was blown with ethyl acetate, 2 mL of 20% methanol was added to the activated C18 column, and 4 mL of 10% methanol was rinsed with 1 mL of 100% methanol (pH 8) to rinse the samples into the bottle. Three independent experiments were performed to ensure reproducibility of the results.

Gene cloning and vector construction

To construct the luciferase reporter gene vector of the LBD38-1 promoter as follows: from −1150 bp to −125 bp, segment 0 was named and amplified by primers F1 and R1 (Supplementary file 7). The −1150 bp to −584 bp segment was named the Q1 segment and amplified via primers F1 and R2. The −604 to −125 bp segment was named the Q2 segment, which was amplified by F2 and R1. The 20 bp segment of the GA element was deleted from the Q1 segment, which was named the Q1* segment and amplified by F3 and R3. The segment 20 bp after the GA element was deleted in Q2 was named the Q2* segment and amplified by F4 and R4.

Specific primers were used to amplify the five promoter fragments (Q0, Q1, Q1*, Q2, and Q2*), and the restriction sites were SalI and BamHI, which were ligated to the pGREEN II 0800-LUC vector.

Functional analysis of the cis-acting elements of the LBD38-1 promoter via a luciferase reporter gene validation assay

The mixture was subsequently transferred to Agrobacterium, which was subsequently used to infect tobacco (Nicotiana benthamiana, 4 weeks of age). Tobacco leaves were treated with 100 mg/L GA3 for GA treatment on one side and untreated with GA on the other side as a control. The fluorescence was observed after 2 h of incubation. The activities of LUC and REN were determined via a dual-luciferase assay system (APE × BIO Technology LLC, Houston, USA), and the relative ratio of LUC/REN was calculated. Each experiment included three biological replicates for each treatment group.

Statistical analysis

The experiments were performed in accordance with a completely randomized design. The data are shown as the means ± standard errors (SEs) of three or six independent biological replicates. The data were analyzed via SPSS (version 20.0; IBM Corp., Armonk, NY, USA) and Excel 2010 software (Microsoft Corp., Redmond, WA, USA).

Data availability

The RNA-Seq datasets are available from the GSA (submission number: CRA011411; https://bigd.big.ac.cn/gsa/browse/CRA011411; submission number: CRA011462; https://bigd.big.ac.cn/gsa/browse/CRA011462; submission number: CRA004704; https://bigd.big.ac.cn/gsa/browse/CRA004704; submission number:CRA017929; https://ngdc.cncb.ac.cn/gsa/browse/CRA017929) and NCBI (https://www.ncbi.nlm.nih.gov/biosample/SAMN25872099).

The CDS and genome sequences of LBDs in walnut were retrieved from the whole walnut genome database (accession GCA_001411555.2) of the NCBI. All the data and materials are presented in the main paper and additional files.

Abbreviations

LBD:

Lateral organ boundary domain

Xaj. :

Xanthomonas arboricola pv. Juglandis

ML:

Mature leaves

YL:

Young leaves

YS:

Young stems

GA:

Gibberellin

NJ:

Neighbor-joining method

References

  1. Wen C, Zhang Z, Cao L, Liu G, Liang L, Liu X, et al. Walnut protein: a rising source of high-quality protein and its updated comprehensive review. J Agric Food Chem. 2023;71(28):10525–42.

    Article  CAS  PubMed  Google Scholar 

  2. Hayes D, Angove MJ, Tucci J, Dennis C. Walnuts (Juglans regia) chemical composition and research in human health. Crit Rev Food Sci Nutr. 2016;56(8):1231–41.

    Article  CAS  PubMed  Google Scholar 

  3. Zhang ZH, Pei D. Walnut science. Beijing: China Agricultural Publishing House; 2018. p. 1–2.

    Google Scholar 

  4. Liu B, Jing D, Liu F, Ma H, Liu X, Peng L. Serendipita indica alleviates drought stress responses in walnut (Juglans regia L.) seedlings by stimulating osmotic adjustment and antioxidant defense system. Appl Microbiol Biotechnol. 2021;105(23):8951–68.

    Article  CAS  PubMed  Google Scholar 

  5. Li D, Peng S, Chen S, Li Z, He Y, Ren B, et al. Identification and characterization of 5 walnut MYB genes in response to drought stress involved in ABA signaling. Physiol Mol Biol Plants. 2021;27(6):1323–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Yang G, Gao X, Ma K, Li D, Jia C, Zhai M, et al. The walnut transcription factor JrGRAS2 contributes to high temperature stress tolerance involving in Dof transcriptional regulation and HSP protein expression. BMC Plant Biol. 2018;18(1):367.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhao B, Liu F, Gong YH, Zhang JP, Ma QG. Walnut blight investigation, spray materials screening, and bactericide application timing for walnut blight control in Northeast China. Eur J Plant Pathol. 2021;161(1):161–70.

    Article  CAS  Google Scholar 

  8. Ma T, Yang C, Cai F, Chen Z. Morpho-cultural, physiological and molecular characterization of Colletotrichum nymphaeae causing anthracnose disease of walnut in China. Microb Pathog. 2022;166: 105537.

    Article  CAS  PubMed  Google Scholar 

  9. Zhang F, Wang J, Ding T, Lin X, Hu H, Ding Z, et al. MYB2 and MYB108 regulate lateral root development by interacting with LBD29 in Arabidopsis thaliana. J Integr Plant Biol. 2024;66(8):1675–87.

    Article  CAS  PubMed  Google Scholar 

  10. Chen WF, Wei XB, Rety S, Huang LY, Liu NN, Dou SX, et al. Structural analysis reveals a “molecular calipers” mechanism for a Lateral organ boundaries domain transcription factor protein from wheat. J Biol Chem. 2019;294(1):142–56.

    Article  CAS  PubMed  Google Scholar 

  11. Majer C, Hochholdinger F. Defining the boundaries: structure and function of LOB domain proteins. Trends Plant Sci. 2011;16(1):47–52.

    Article  CAS  PubMed  Google Scholar 

  12. Ye L, Wang X, Lyu M, Siligato R, Eswaran G, Vainio L, et al. Cytokinins initiate secondary growth in the Arabidopsis root through a set of LBD genes. Curr Biol. 2021;31(15):3365–73: e7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang YM, Zhang SZ, Zheng CC. Genomewide analysis of lateral organ boundaries domain gene family in Zea mays. J Genet. 2014;93(1):79–91.

    Article  CAS  PubMed  Google Scholar 

  14. Wang Z, Zhang R, Cheng Y, Lei P, Song W, Zheng W, et al. Genome-wide identification, evolution, and expression analysis of LBD transcription factor family in bread wheat (Triticum aestivum L.). Front Plant Sci. 2021;12:721253.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Xu J, Hu P, Tao Y, Song P, Gao H, Guan Y. Genome-wide identification and characterization of the lateral organ boundaries domain (LBD) gene family in polyploid wheat and related species. PeerJ. 2021;9: e11811.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Liu H, Cao M, Chen X, Ye M, Zhao P, Nan Y, et al. Genome-wide analysis of the lateral organ boundaries domain (LBD) gene family in solanum tuberosum. Int J Mol Sci. 2019;20(21): 5360.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Cao H, Liu CY, Liu CX, Zhao YL, Xu RR. Genome-wide analysis of the lateral organ boundaries domain gene family in Vitis vinifera. J Genet. 2016;95(3):515–26.

    Article  CAS  PubMed  Google Scholar 

  18. Han Z, Yang T, Guo Y, Cui WH, Yao LJ, Li G, et al. The transcription factor PagLBD3 contributes to the regulation of secondary growth in Populus. J Exp Bot. 2021;72(20):7092–106.

    Article  CAS  PubMed  Google Scholar 

  19. Liu L, Zhang J, Xu J, Li Y, Lv H, Wang F, et al. SlMYC2 promotes SlLBD40-mediated cell expansion in tomato fruit development. Plant J. 2024;118(6):1872–88.

    Article  CAS  PubMed  Google Scholar 

  20. Yu J, Gao B, Li D, Li S, Chiang VL, Li W, et al. Ectopic expression of PtrLBD39 retarded primary and secondary growth in Populus trichocarpa. Int J Mol Sci. 2024;25(4):2205.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhang Y, Li Y, de Zeeuw T, Duijts K, Kawa D, Lamers J, et al. Root branching under high salinity requires auxin-independent modulation of lateral organ boundary domain 16 function. Plant Cell. 2024;36(4):899–918.

    Article  PubMed  Google Scholar 

  22. Zheng L, Chao Y, Wang Y, Xu Y, Li S. Genome-wide analysis of the LBD gene family in melon and expression analysis in response to wilt disease infection. Genes (Basel). 2024;15(4):442.

    Article  CAS  PubMed  Google Scholar 

  23. Liu W, Yu J, Ge Y, Qin P, Xu L. Pivotal role of LBD16 in root and root-like organ initiation. Cellular and molecular life sciences. Cell Mol Life Sci. 2018;75(18):3329–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lee HW, Cho C, Pandey SK, Park Y, Kim MJ, Kim J. LBD16 and LBD18 acting downstream of ARF7 and ARF19 are involved in adventitious root formation in Arabidopsis. BMC Plant Biol. 2019;19(1):46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gupta K, Gupta S. Molecular and in silico characterization of tomato LBD transcription factors reveals their role in fruit development and stress responses. Plant Gene. 2021;27(4): 100309.

    Article  CAS  Google Scholar 

  26. Song B, Tang Z, Li X, Li J, Zhang M, Zhao K, et al. Mining and evolution analysis of lateral organ boundaries domain (LBD) genes in Chinese white pear (Pyrus bretschneideri). BMC Genomics. 2020;21(1):644.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Li HH, Liu X, An JP, Hao YJ, Wang XF, You CX. Cloning and elucidation of the functional role of apple MdLBD13 in anthocyanin biosynthesis and nitrate assimilation. Plant Cell Tiss Org. 2017;130:47–59.

    Article  CAS  Google Scholar 

  28. Liu L, Zhang J, Xu J, Li Y, Guo L, Wang Z, et al. CRISPR/Cas9 targeted mutagenesis of SlLBD40, a lateral organ boundaries domain transcription factor, enhances drought tolerance in tomato. Plant Sci. 2020;301: 110683.

    Article  CAS  PubMed  Google Scholar 

  29. Thatcher LF, Powell JJ, Aitken EA, Kazan K, Manners JM. The lateral organ boundaries domain transcription factor LBD20 functions in fusarium wilt susceptibility and jasmonate signaling in Arabidopsis. Plant Physiol. 2012;160(1):407–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Hu Y, Zhang J, Jia H, Sosso D, Li T, Frommer WB, et al. Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease. Proc Natl Acad Sci USA. 2014;111(4):E521–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Feng X, Xiong J, Zhang W, Guan H, Zheng D, Xiong H, et al. ZmLBD5, a class-II LBD gene, negatively regulates drought tolerance by impairing abscisic acid synthesis. Plant J. 2022;112(6):1364–76.

    Article  CAS  PubMed  Google Scholar 

  32. Wu M, Wang Y, Zhang S, Xiang Y. A LBD transcription factor from moso bamboo, PheLBD12, regulates plant height in transgenic rice. Plant Mol Biol. 2024;114(5):95.

    Article  CAS  PubMed  Google Scholar 

  33. Colebrook EH, Thomas SG, Phillips AL, Hedden P. The role of gibberellin signalling in plant responses to abiotic stress. J Exp Biol. 2014;217(Pt 1):67–75.

    Article  CAS  PubMed  Google Scholar 

  34. Ma W, Pang Z, Huang X, Xu J, Pandey SS, Li J, et al. Citrus Huanglongbing is a pathogen-triggered immune disease that can be mitigated with antioxidants and gibberellin. Nat Commun. 2022;13(1):529.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Gaion LA, Monteiro CC, Cruz FJR, Rossatto DR, López-Díaz I, Carrera E, et al. Constitutive gibberellin response in grafted tomato modulates root-to-shoot signaling under drought stress. J Plant Physiol. 2018;221:11–21.

    Article  CAS  PubMed  Google Scholar 

  36. Sun TP. Gibberellin-GID1-DELLA: a pivotal regulatory module for plant growth and development. Plant Physiol. 2010;154(2):567–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ko JH, Yang SH, Han KH. Upregulation of an Arabidopsis RING-H2 gene, XERICO, confers drought tolerance through increased abscisic acid biosynthesis. Plant J. 2006;47(3):343–55.

    Article  CAS  PubMed  Google Scholar 

  38. Shan C, Mei Z, Duan J, Chen H, Feng H, Cai W. OsGA2ox5, a gibberellin metabolism enzyme, is involved in plant growth, the root gravity response and salt stress. PLoS One. 2014;9(1): e87110.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Wang F, Wang C, Yan Y, Jia H, Guo X. Overexpression of cotton GhMPK11 decreases disease resistance through the gibberellin signaling pathway in transgenic Nicotiana benthamiana. Front Plant Sci. 2016;7:689.

    PubMed  PubMed Central  Google Scholar 

  40. Yang DL, Li Q, Deng YW, Lou YG, Wang MY, Zhou GX, et al. Altered disease development in the eui mutants and Eui overexpressors indicates that gibberellins negatively regulate rice basal disease resistance. Mol Plant. 2008;1(3):528–37.

    Article  CAS  PubMed  Google Scholar 

  41. Dong L, Manghwar H. Genome-wide expression analysis of LBD genes in tomato (Solanum lycopersicum L.) under different light conditions. Plant Signal Behav. 2023;18(1):2290414.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Huang B, Huang Z, Ma R, Ramakrishnan M, Chen J, Zhang Z, et al. Genome-wide identification and expression analysis of LBD transcription factor genes in Moso bamboo (Phyllostachys edulis). BMC Plant Biol. 2021;21(1):296.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wang RR, Bai TH, Gao HY, Cui YJ, Zhou RL, Wang ZY, et al. Genome-wide identification of LBD transcription factors in apple and the function of MdLBD16a in adventitious rooting and callus development. Sci Hortic. 2023;317: 112048.

    Article  CAS  Google Scholar 

  44. Wang J, Zhang W, Cheng Y, Feng L. Genome-wide identification of LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factors and screening of salt stress candidates of Rosa rugosa Thunb. Biology (Basel). 2021;10(10):992.

    PubMed  Google Scholar 

  45. Moore RC, Purugganan MD. The early stages of duplicate gene evolution. Proc Natl Acad Sci USA. 2003;100(26):15682–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Cannon SB, Mitra A, Baumgarten A, Young ND, May G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004;4: 10.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Tian Y, Han X, Qu Y, Zhang Y, Rong H, Wu K, et al. Genome-wide identification of the ginkgo (Ginkgo biloba L.) LBD transcription factor gene and characterization of its expression. Int J Mol Sci. 2022;23(10): 5474.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Xing GW, Wang MX, Ma XF, Zhao X, Zhang T, Nie XJ, et al. Genome-wide analysis, expression characteristics and regulatory network of LBD gene family in bread wheat (Triticum aestivum L.). J Triticeae Crops. 2017;37(7):855–63.

    Google Scholar 

  49. Wu T, Cheng C, Zhong YZG. Molecular characterization of the gibberellin-stimulated transcript of GASA4 in citrus. Plant Growth Regul. 2020;91(1):89–99.

    Article  CAS  Google Scholar 

  50. Zhang X, He Y, He W, Su H, Wang Y, Hong G, et al. Structural and functional insights into the LBD family involved in abiotic stress and flavonoid synthases in Camellia sinensis. Sci Rep. 2019;9(1):15651.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Weston DJ, Karve AA, Gunter LE, Jawdy SS, Yang X, Allen SM, et al. Comparative physiology and transcriptional networks underlying the heat shock response in Populus trichocarpa, Arabidopsis thaliana and Glycine max. Plant Cell Environ. 2011;34(9):1488–506.

    Article  CAS  PubMed  Google Scholar 

  52. Chen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, et al. TBtools-II: a “one for all, all for one” bioinformatics platform for biological big-data mining. Mol Plant. 2023;16(11):1733–42.

    Article  CAS  PubMed  Google Scholar 

  53. Song XB, Chang YY, Liu H, Xu HM, Pei D. Reference gene selection and genes expression analysis during adventitious root formation in walnut. Acta Hortic Sinica. 2019;46(10):1907–18.

    Google Scholar 

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Acknowledgements

We would also like to thank Dr. Huibin Wang for valuable suggestions on the data analysis and manuscript.

Funding

This work was supported by the Natural Science Foundation of Hebei Province (C2023204269 and C2024204128). Funds were used for the design of the study; sample collection, analysis or interpretation of the data; and writing of the manuscript.

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Author notes

  1. Wenqiang Dai, Yaoling Li and Zhenghan Chen contributed equally to the work.

Authors and Affiliations

  1. College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China

    Wenqiang Dai, Yaoling Li, Zhenghan Chen, Hui Wang & Shugang Zhao

  2. College of Biochemical and Environmental Engineering, Baoding University, Baoding, 071001, China

    Fuqiang He

  3. College of Horticulture, Hebei Agricultural University, Baoding, 071001, China

    Jiali Peng, Hongxia Wang & Xiuhong An

  4. College of Biological Engineering, Binzhou Polytechnic, Binzhou, 256603, China

    Kai Liu

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Contributions

S.Z, W.D and Y.L designed the research. S.Z, W.D, Y.L, and Z.C performed the experiments, analyzed the data and wrote the paper. H.W, J.P, F.H, K.L, H.W and X.A participated in the data analysis. All authors read and approved the final the manuscript.

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Correspondence to Shugang Zhao.

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Walnut is a widespread fruit tree in China and is not an endangered species. Walnut trees were obtained from the Experimental Field of Hebei Agricultural University. No specific permits were required for sample collection from the walnuts.

All the plant materials used followed national and international standards and local laws and regulations. The use of plant materials does not pose any risk to other species. No specific permission was required to collect all the samples described in this study.

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Dai, W., Li, Y., Chen, Z. et al. Gibberellin Regulates LBD38-1 Responses to Xanthomonas arboricola pv. juglandis Infection in Walnut Bacterial Blight Pathogenesis. BMC Genomics 26, 370 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11518-9

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Keywords

BMC Genomics

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