LOG protein sequences from Arabidopsis were downloaded from the TAIR database and utilized as query sequences to conduct a BLAST search against the protein-coding genes in the walnut genome sequenced by Huang Xuehui’s Lab (http://www.xhhuanglab.cn/ (accessed on 20 November 2024)), with an E-value threshold set at 1E−5. The obtained sequences were further examined using Pfam (http://pfam-legacy.xfam.org/ (accessed on 20 November 2024)) and CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 20 November 2024)) to verify the presence of specific domains. The identified candidate genes were renamed according to their chromosomal locations. The physicochemical properties of these walnut LOG (JrLOG) proteins were examined online using the ExPASy (https://www.expasy.org/ (accessed on 20 November 2024)).

The LOG protein sequences from rice, Arabidopsis, and walnut were used to perform a multiple sequence alignment analysis. The resulting alignment was used to construct a phylogenetic tree using MEGA7.0 software, employing the Neighbor-Joining (N-J) statistical method with 1000 bootstrap replicates and the Gamma distribution model. Based on genome annotation data, the chromosomal locations of each JrLOG gene were visualized using TBtools software.

The gene structures of JrLOG genes, including coding sequences and introns, were visualized using the GSDS online tool (https://gsds.cgrpoee.top/ (accessed on 20 November 2024)). Conserved motifs in the JrLOG proteins were detected using the MEME suite (https://meme-suite.org/meme/ (accessed on 20 November 2024)) (Table S1).

Collinearity relationships among walnut chromosomes, as well as between walnut, Arabidopsis, and rice, were analyzed using the MCScanX program. Gene duplication events for JrLOG in walnut were visualized with Advanced Circos. The nonsynonymous (Ka) and synonymous (Ks) substitution rates, along with their ratio (Ka/Ks), were calculated using the KaKs Calculator software (Table S2).

The 1500 bp upstream sequences from the start codon of each JrLOG gene were extracted from the walnut genome and submitted to the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 20 November 2024)) for the prediction of cis-regulatory elements. The number and distribution of these elements were visualized after statistical analysis.

Two-year-old, uniformly growing Juglans regia ‘Lvling’ walnut trees were used as experimental materials and randomly divided into two groups. For cold treatments, one group was placed in a growth chamber at 4°C, while the control group was kept at 25°C. Walnut leaves were collected at 0.5, 1, and 2 h post-treatment, immediately frozen in liquid nitrogen and stored at −80°C.

The expression profiles of JrLOG genes in various walnut tissues were based on RNA-Seq data from 19 different tissues [23]. The expression patterns of JrLOG in walnut endopleura during different developmental stages were derived from transcriptomic data covering 35 to 147 days post-flowering [24]. The expression levels of JrLOG in response to Colletotrichum gloeosporioides infection were analyzed using published RNA-Seq data [25]. The response of JrLOG to cold stress was further explored using quantitative real-time PCR (qPCR) following the protocol described by Zheng et al. [26]. The primers used are listed in Table S1.

The coding sequence of JrLOG3 was cloned from walnut leaf cDNA following the method outlined by Jia et al. [27]. The predicted amino acid sequence was employed for multiple sequence alignment and phylogenetic analysis, as described in Section 2.2, by comparing it with LOG proteins from different species with known functions. The JrLOG3 coding region was inserted into the pCambia2300 vector, creating a JrLOG3-EGFP expression cassette driven by the 35S constitutive promoter. The recombinant vector was then introduced into tobacco (Nicotiana benthamiana) epidermal cells via Agrobacterium-mediated transformation. After 3 days of dark incubation, the transformed tobacco cells were examined for green fluorescent protein (GFP) signals using confocal microscopy, following the previously described procedure [26].

Nine AtLOG genes were identified and documented in the Arabidopsis thaliana genome. BLASTP searches were performed against the walnut genome protein database to discover LOG genes using the nine AtLOG protein sequences. Seventeen potential JrLOG genes were identified by manual inspection and verification using the NCBI conserved domain database (Table 1). These JrLOG genes were designated sequentially based on their chromosomal locations (JrLOG01–JrLOG17). The 17 JrLOG genes were distributed across 11 chromosomes of the walnut genome, with chromosome 13 containing the highest number of LOG genes (3), while chromosomes 8, 9, 10, and 14 each contained two genes. Chromosomes 5, 6, 7, 12, 15, and 16 each housed a single JrLOG gene (Table 1).

The relationships among the LOG genes were elucidated by constructing a neighbor-joining tree based on the LOG protein sequences from Arabidopsis, rice, and walnut. According to the phylogenetic tree (Fig. 1), these proteins were classified into six groups, labeled Group I to Group VI, with all walnut LOG proteins clustering into Groups I to V.

The lengths of JrLOG genes ranged from 1326 to 4317 bp. All members of the JrLOG gene family contained 4 to 6 introns. Specifically, JrLOG10 had 4 introns, JrLOG4 contained 5, while the remaining members had 6 introns. The number and distribution of introns were relatively conserved within each phylogenetic cluster (Fig. 2A,B). For instance, the introns in Group I genes were notably short, while those in Group IV were much longer. This conservation of gene structure within evolutionary clades suggested that these genes have been subjected to similar evolutionary pressures.

Physicochemical property analysis of the JrLOG proteins revealed that their amino acid lengths ranged from 157 to 302, with estimated molecular weights between 17.13 and 33.15 kDa. The theoretical isoelectric points (pI) ranged from 5.04 to 8.61, encompassing both acidic and basic values. The instability index of these proteins varied between 30 and 50, with more than half of the members exhibiting an index greater than 40, indicating that JrLOG proteins were generally unstable (Table 1).

MEME analysis indicated that, except JrLOG10 and JrLOG4, all members possessed three characteristic motifs. JrLOG4 contains two specific structural domains, while JrLOG10 had only one (Fig. 2C). Notably, protein domain analysis further revealed that all members contained a conserved PpnN domain, a core component of nucleotide monophosphate nucleosidases (Fig. 2D). Multiple sequence alignment confirmed that the amino acid sequences within these three motifs were highly conserved, except in JrLOG10 and JrLOG4 (Fig. 2E).

Gene collinearity analysis was performed to uncover the conservation and evolutionary patterns of gene family members across different species. Although JrLOG members were distributed across various chromosomes in differing numbers (Fig. 3A), nine pairs of collinear genes were detected among JrLOG members in walnut (Fig. 3B). Except for JrLOG10, all other members exhibited collinear relationships. Notably, these collinear genes were not located on the same chromosome. Additionally, collinearity relationships were observed among the three Group I members, JrLOG01, JrLOG02, and JrLOG05.

Thirteen pairs of collinear LOG genes were identified between walnut and Arabidopsis (Fig. 3C), while only seven collinear pairs were detected between walnut and rice (Fig. 3D). This suggests that LOG genes in walnut share a closer evolutionary relationship with those in Arabidopsis than with rice.

Cis-regulatory elements (CREs) play a critical role in understanding gene regulation, phenotype expression, and plant adaptability. To gain insights into the regulatory mechanisms of JrLOG genes, we analyzed the potential CREs in their promoter regions (Fig. 4).

Several elements associated with plant hormone responses were identified, including those responsive to ABA (ABRE), auxin (TGA-element), MeJA (TGACG-motif and CGTCA-motif), GA (P-box and TATC-box), and SA (TCA-element), which were present in many members of the gene family. Notably, all members, except JrLOG6, contained ABA-responsive elements, with JrLOG3 exhibiting the highest number of ABRE motifs. Interestingly, auxin-responsive elements were found exclusively in the JrLOG6 promoter. Additionally, tissue-specific expression elements were identified, such as seed-specific expression elements (RY-element) in JrLOG08 and JrLOG14, and meristem-specific elements (CAT-box) in JrLOG01. CREs related to developmental processes were also detected, including two circadian control elements in JrLOG08 and cell cycle-related MSA-like elements in JrLOG16. Moreover, several CREs associated with environmental responses were predicted, such as those related to low temperature (LTR), drought (MBS), and light response (ACE-element). Interestingly, the distribution of CREs was not conserved among members of the same evolutionary clade, suggesting that JrLOG genes may be involved in a diverse array of biological processes.

Tissue-specific expression analysis (Fig. 5A) revealed that JrLOG3, JrLOG10, JrLOG11, and JrLOG16 were highly expressed in the leaf mature (LM), leaves (LE), and leaf young (LY). JrLOG04 exhibited notably high expression in the pistillate flower (FL), while JrLOG07 showed strong expression exclusively in vegetative buds (VB). JrLOG01 and JrLOG09 were highly expressed in hull dehiscing (HU) and hull peel (HP), respectively, whereas JrLOG13 had high expression in hull immature (HL). Additionally, JrLOG08 and JrLOG17 were strongly expressed in catkins (CK). JrLOG12 was the only gene with high expression in both the callus exterior (CE) and callus interior (CI). JrLOG02 and JrLOG06 exhibited elevated expression levels in somatic embryo (SE), while JrLOG05 and JrLOG14 were specifically highly expressed in roots (RT).

The walnut endopleura stores nutrients, protects the seed, and influences germination. Therefore, the expression profiles of JrLOG genes during seed endopleura development were also analyzed (Fig. 5B). JrLOG01, JrLOG08, JrLOG11, JrLOG13, and JrLOG15 displayed high expression during the early stages of endopleura development, whereas the remaining members exhibited increased expression closer to fruit maturation. Notably, JrLOG3 was significantly upregulated after the fruit hardening stage.

Upon inoculation with anthracnose, the expression levels of JrLOG genes fluctuated noticeably (Fig. 6). JrLOG05 was upregulated in genotype F423 genotype only 48 h post-infection, whereas, in the F26 genotype, its upregulation was delayed until 72 h and showed only a slight increase. In contrast, JrLOG10 in F26 exhibited two distinct expression peaks at 48 and 120 h post-infection, whereas in F423, it exhibited a consistently low level of expression. Additionally, JrLOG17 in F26 exhibited significant induction at both 24 and 48 h post-infection compared to F423. Notably, despite a general downregulation in transcription, JrLOG3 maintained relatively high expression in F26 at 24 h post-infection compared to its expression in F423. These findings suggest potential differences in the stress response mechanisms between the two genotypes.

Under cold stress, the expression of JrLOG gene family members exhibited substantial variations (Fig. 7). JrLOG5, JrLOG8, JrLOG14, and JrLOG15 appeared to be unresponsive to cold treatment, while JrLOG6, JrLOG12, and JrLOG13 showed a general tendency towards downregulation in their expression levels after cold treatment, despite also experiencing fluctuations. Additionally, some genes displayed fluctuating expression patterns without any specific trend, including JrLOG6, JrLOG9, JrLOG10, JrLOG16, and JrLOG17. No significant differences in transcription levels were detected for JrLOG1, JrLOG2, JrLOG4, and JrLOG7 at 0.5 h and 1 h post-cold treatment; however, significant induction in expression was observed at 2 h. Notably, JrLOG3 and JrLOG11 exhibited strong transcriptional activation as early as 0.5 h post-cold treatment and were induced at all sampling time points.

Using leaf tissue as the material, the expected band size for JrLOG3 was successfully amplified through via transcription PCR (Fig. 8A). Sequencing confirmed that the coding region of JrLOG3 is 678 bp in length, encoding a 225-amino-acid protein with a predicted molecular weight of 25.4 kDa. Phylogenetic analysis indicated that JrLOG3 was closely related to LOG proteins from other species, notably GhLOG3 from cotton and SlycLOG1a from tomato (Fig. 8B). Multiple sequence alignment demonstrated that JrLOG3 shares a high degree of conservation with these LOG proteins, including the catalytic sequence ‘PGGxGTxE’, a key feature situated at the core of the protein’s three-dimensional structure. This catalytic motif was entirely conserved across all analyzed proteins (Fig. 8C,D). Subcellular localization analysis revealed that GFP fluorescence driven by JrLOG3 was exclusively expressed in the cell membrane (Fig. 8E).

LOG genes have been identified in several plant species; however, the LOG gene family in walnut (Juglans regia), an economically important woody oil tree species, has not been systematically explored. In this study, we identified 17 LOG-encoding genes in the walnut genome through homology-based comparisons. The JrLOG members were categorized into five subgroups using phylogenetic analysis (Fig. 1 and Fig. 2A). It appears that these genes have undergone duplication events (Fig. 3), likely associated with a whole-genome duplication approximately 20 million years ago [28]. Consequently, As a result, JrLOG genes are located on several chromosomes, comprising nine pairs of syntenic genes (Fig. 3B). Similarly, the expansion of the RcLOG gene family in castor bean (Ricinus communis) was driven by gene duplication events [19]. In walnut, this expansion was accompanied by purifying selection, as indicated by the Ka/Ks ratios of all syntenic gene pairs being much less than 1 (Table S2). Closely grouped JrLOG genes display similar gene architectures, amino acid sequences, conserved motifs, and domain characteristics (Fig. 2). Nonetheless, JrLOG10 lacks synteny with other members, suggesting it may have evolved as an independent lineage, leading to the loss of at least three exons and the absence of two characteristic motifs.

Moreover, closely related members, such as JrLOG3 and JrLOG4 (Fig. 2A), although forming a pair of syntenic genes (Fig. 3B), exhibited exon loss during gene duplication (Fig. 2B). This loss resulted in JrLOG4 lacking a C-terminal motif (Fig. 2C) and retaining only a diminished PpnN domain (Fig. 2D). Similarly, some members of the KNOX gene family have evolved without the C-terminal domain, forming distinct clades with functions similar to those of other members [29]. Notably, even closely related JrLOG genes displayed variation in the number and types of CREs in their promoters (Fig. 4), potentially explaining the balance between conservation and variability within the JrLOG family. Thirteen pairs of syntenic LOG genes were identified between walnut and Arabidopsis (Fig. 3C), whereas only seven pairs were found between walnut and rice (Fig. 3D), likely due to the closer evolutionary relationship among dicotyledonous plants compared to that between dicots and monocots.

The LOG gene family plays a critical role in plant growth and development [12]. Thus, we systematically characterized the expression profiles of the JrLOG genes across various tissues and developmental stages. The abundance of JrLOG3, JrLOG10, JrLOG11, and JrLOG16 at various phases of leaf development indicates that these genes are essential for this process. Their expression may enhance endogenous CK levels, which could improve leaf size and regulate plant architecture [30]. Its possible function in floral development and reproduction was indicated by the specific and strong expression of JrLOG04 in pistillate flowers, an organ that is essential for walnut output. These results are in agreement with those from Arabidopsis, whose LOG genes control the development of floral meristems and are expressed in flowers [12]. In rice, LOG1 influences CK levels in young panicles, which modulates grain yield [11].

Seed coats, essential for seed germination, respond positively to exogenous CK treatments [31]. The high expression of JrLOG1, JrLOG9, and JrLOG13 in the hull suggests their possible involvement in hormone regulation related to seed germination [32]. CK application has been shown to enhance shoot regeneration efficiency in peanuts (Arachis hypogaea L.) [33] and to support callus growth by elevating endogenous CK levels [34]. JrLOG12 was specifically expressed in callus tissue, implicating it in tissue regeneration and cellular reprogramming [35]. Additionally, in Medicago truncatula, MtLOG1 and MtLOG2 regulate lateral root formation [36], raising the possibility that the root-specific expression of JrLOG5 and JrLOG14 in walnut may serve a similar function.

CKs also play a critical role in plant immunity against pathogens. For instance, infection by Verticillium longisporum alters CK levels, while in legumes, CK responses vary following bacterial inoculation [37]. In disease-resistant walnut germplasm, JrLOG3 maintained high expression 24 h after pathogen infection, suggesting its potential role in walnut disease resistance (Fig. 6). Under cold stress, JrLOG3 and JrLOG11 were upregulated, with JrLOG03 exhibiting rapid and continuous activation (Fig. 7). Conversely, several JrLOG genes were downregulated and displayed fluctuating expression patterns under cold stress, possibly as a strategy to slow growth in adverse conditions [38]. Given the significant expression of JrLOG3 in leaves and its responsiveness to both biotic and abiotic stresses, we cloned and analyzed this gene further. The cloned JrLOG3 is closely related to GhLOG3 from cotton (Fig. 8B), which induces adversity stress resistance [20]. While transient and stable expression of LOG fusion proteins in rice, Arabidopsis, and Chlamydomonas reinhardtii indicated localization in the cytoplasm and nucleus [9], JrLOG3 exhibited subcellular localization to the cell membrane (Fig. 8E). Similar to other LOG proteins, JrLOG3 contains the conserved “PGGxGTxxE” sequence, which contributes to AMP stability, indicating a shared catalytic mechanism. Consequently, JrLOG3 is a strong candidate for further investigation into its role in cytokinin regulation, organ development (e.g., leaf expansion), and stress tolerance.