Genome-wide characterization and expression analysis of the CONSTANS-like gene family of Juglans mandshurica Maxim

Identification of JmCOL genes in Juglans mandshurica

In the phylogenetic tree, the protein sequences of the CONSTANS (CO) gene family in A. thaliana were obtained through TAIR (https://www.arabidopsis.org/) (Yang et al., 2024). A. thaliana CO protein sequences were used to search the J. mandshurica genome database using the blastx program in BLAST (blast-2.6.0+) with E-value < 10⁻¹⁰. The HMM (Hidden Markov Model) of COLs was constructed by adopting HMMER 3.3.2 (http://www.hmmer.org/download.html). The results of BLAST and HMMER were merged, and the incomplete or redundant sequences were eliminated. A total of 15 JmCOL genes with both B-BOX and CCT conserved domains of J. mandshurica were identified. In addition, the information and various physicochemical parameters of JmCOL genes were analyzed using ProtParam (https://web.expasy.org/protparam/) (Yang et al., 2024).

Chromosomal localization of JmCOLs genes, analysis of gene structure and conserved domains

J. mandshurica exons and introns of JmCOL genes were displayed using GSDS2.0 (http://gsds.gao-lab.org/). Conserved motifs in JmCOL were analyzed using MEME software (https://meme-suite.org/meme/tools/tomtom) with default parameters, as previously described (Yang et al., 2024). The JmCOL gene was localized on chromosomes using TBtools-II (Toolbox for Biologists) version 2.136. The conserved domains identified through MEME analysis were also marked using TBtools-II (Toolbox for Biologists) version 2.136.

JmCOLs phylogenetic analysis

Phylogenetic analyses were generated with MEGA7.0.26 software. Sequence alignment of proteins from two species (J. mandshurica, A. thaliana) was performed with MUSCLE, and a phylogenetic tree was constructed with the Neighbor-Joining (NJ) tree, Bootstrap replications = 5 00, Method is p-distance model, Site Coverage Cutoff 50%. The phylogenetic tree constructed by MEGA 7.0.26 was uploaded to iTOL (https://itol.embl.de/) for further editing.

Collinearity analysis

Genome data of J. mandshurica and A. thaliana were used to analyze their collinearity relationships. TBtools-II v2.136 software was used to calculate and draw the whole gene collinearity analysis of species.

Cis-acting elements analysis

The 2 kb DNA sequence upstream from the ATG of JmCOL genes was extracted from the J. mandshurica genome database (https://cmb.bnu.edu.cn/juglans/) as the promoter region. Cis-elements were analyzed in the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Analysis results were visualized with TBtools-II v2.136 software.

Real-time quantitative PCR (RT-qPCR) analysis

Total RNA was extracted from various organs and tissues of the J. mandshurica using a plant RNA extraction mini kit. Its integrity and concentration were then assessed through agarose gel electrophoresis and measured with a Thermo NanoDrop 2000 instrument; cDNA was synthesized using HyperScriptTM III RT SuperMix for qPCR with gDNA Remover to synthesize the first strand of cDNA. Quantitative primers were designed using PrimerPremier6 software, and primer specificity was verified using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) from NCBI, with 18S as the internal reference gene. The instrument used for Real-time fluorescence quantitative PCR is from Analytik Jena, and its name and model are 3107B - 0545 and qTOWER3/G respectively. The reaction system for quantitative PCR has a volume of 20 µL. In this system, the quantitative enzyme used is Taq Pro Universal SYBR qPCR Master Mix. The total amount of cDNA in the reaction volume is 0.1–1 µM. The forward and reverse quantitative primers each have a volume of 0.4 µL (10 µM). Finally, ddH₂O is added to make up the volume to 20 µL. The instruments and the reaction procedures used are detailed in Table S2.

Plant materials

On April 2, 2022, in the natural mother forest of Liao Ning province experimental forestry field, Liaoning, China (124.9°E, 42.13°N), healthy protogynous and protandrous type of J. mandshurica were chosen. At this time, J. mandshurica buds are at the stage of physiological differentiation, that is, the period when flower buds are just produced as shown in Figs. 1A–1B. Four bud types were collected with three replicate samples for each group of bud types and numbered one by one. The samples in group T1 were protogynous female buds named CC (A1, A2, A3), the samples in group T2 were protogynous male buds named CX (A4, A5, A6), the samples in group T3 were protandrous female buds named XC (B1, B2, B3), and the samples in group T4 were protandrous male buds named XX (B4, B5, B6). The total number of samples was 12. Flower buds were rapidly frozen in liquid nitrogen and stored at −80 °C. A total of 12 RNA seq libraries (A1-6, B1-6) were constructed. The collected samples were sent to Paisano Biotechnology Ltd (Shanghai, China) for high-throughput sequencing of the cDNA libraries. The project number is YFnj20201682. The sequencing data were filtered using Cut adapt to remove any junctions at the 3′ end and any reads with an average mass fraction lower than Q20, in order to obtain clean reads for subsequent analysis and identification.

Starting from April 2, 2024, samples were collected from ten different developmental periods: April 2 (4.2), April 10 (4.10), April 14 (4.14), April 24 (4.24), April 30 (4.30), May 4 (5.4), May 13 (5.13), May 17 (5.17), May 20 (5.20), and May 22 (5.22). The period from April 2 to April 30 was characterized by physiological differentiation, while the latter half of this period was marked by morphological differentiation. Protogynous and protandrous type flower buds from the natural seed forest of J. mandshurica at the experimental forestry farm in Liaoning Province. Four different types of flower buds were collected (Figs. 1A–1F) with each sample containing three independent biological replicates. The number of samples per period was 12 (each of the four types of buds contained three biological replicates). The total number of all samples was 120, and these flower bud samples were quickly frozen in liquid nitrogen after collection and stored at −80 °C refrigerator.

On June 2, 2024, at the experimental base of Shenyang Agricultural University, an appropriate quantity of samples was carefully collected from the leaves, flower buds, stems, and fruits of J. mandshurica. Each sample type underwent three biological replicates, summing up to a total of 12 samples. Post-collection, these samples were promptly wrapped in aluminum foil and plunged into liquid nitrogen for rapid freezing. Subsequently, they were transferred and stored in an ultra-low-temperature refrigerator set at −80 °C for future experimental requirements.

Data and statistical analysis

The J. mandshurica genome database (Genome Assembly V1.3) was obtained from the website (https://cmb.bnu.edu.cn/juglans/) (Mortazavi et al., 2008). To facilitate the comparability of gene expression levels across genes and samples, the expression amounts were normalized using FPKM (Fragments Per Kilobase of transcript per Million map pe dreads) (Trapnell et al., 2010). FPKM is calculated as the number of fragments per kilobase of gene length per million mapped fragments, based on the alignment results. The heatmap was plotted by https://www.bioinformatics.com.cn (last accessed on 10 Oct 2024), an online platform for data analysis and visualization (Tang et al., 2023).

All experimental data included at least three biological replicates. The tissue-specific expression maps of JmCOLs gene were performed using Excel software (Microsoft Office, 2021). Bar charts were determined by one-way ANOVA (* stands for p < 0.05, 0.01). The relative expression of nine JmCOL genes was calculated by 2^−ΔΔCT method (Livak & Schmittgen, 2001), and the results were subjected to one-way ANOVA (* stands for p < 0.05, 0.01) using SPSS 26. Post hoc multiple tests were performed on the data results, assuming that the equal variance was Duncan H. The tissue-specific expression maps of JmCOLs gene were performed using Microsoft Excel (Microsoft, Inc., Redmond, WA, USA).

Identification and analysis of the JmCOLs gene family in Juglans mandshurica

The structural domain integrity of the candidate members was confirmed using NCBI Batch CD-search and the MEME website (https://meme-suite.org/meme/tools/tomtom). Ultimately, the protein sequences encoded by 15 JmCOL genes were obtained, and each gene was named according to its level of homology to A. thaliana CO and COLs (Table 1). It was observed that the full length of the CDS sequences of the JmCOLs genes ranged from 1,026 bp to 1,557 bp, encoded 341–518 amino acids, and had molecular weights of 37.57–52.5 kDa. Additionally, the theoretical isoelectric point ranged from 5.18 to 6.68, indicating that all of the JmCOL proteins were acidic. Subcellular localization prediction revealed that the majority of JmCOL proteins are localized to the nucleus, while the remaining members are localized to the Golgi apparatus.

The chromosomal localization of JmCOL genes was conducted, revealing that 15 of these genes are distributed across nine chromosomes of the J. mandshurica genome. This distribution is illustrated in Fig. 2. For example, JmCOL 2, JmCOL12, and JmCOL9a were located on chromosome 3; JmCOL 13, JmCOL5, and JmCOL4a were located on chromosome 7; and JmCOL11, JmCOL4b, JmCOL16b, JmCOL6, and JmCOL16a were located on chromosomes 4, 8, 9, 16, and 15, respectively. Chromosome 5 and chromosome 6 each contain two genes.

Phylogenetic analysis of the JmCOLs gene family in Juglans mandshurica

Phylogenetic and structural diversity play a key role in the evolutionary relationships of gene families. COL protein sequences were downloaded from the A. thaliana database to construct a phylogenetic tree of COL proteins in nutmeg and A. thaliana (Fig. 3). The CO family can be divided into three main taxa in A. thaliana: Taxon 1 includes CO and COL1∼COL5 with two B-boxes and six conserved amino acids known as T motifs at their carboxyl termini; taxon 2 includes COL6∼COL8 and COL16 that have one B-box; and Taxon 3 includes COL9∼COL15, with one B-box and a second divergent zinc finger structural domain (Hassidim et al., 2009). Based on the evolutionary tree, JmCOL proteins can be classified into three groups (Group I, Group II and Group III), where Group III contains the most JmCOL family members with eight, followed by Group I with four and Group II with the least number of three members.

Conserved motif and gene structure analysis of the JmCOLs protein family in Juglans mandshurica

To further investigate the structural diversity of JmCOL genes, we compared the number and composition of exons and introns of JmCOLs genes, as well as the corresponding evolutionary relationships (Fig. 4A), and the results were similar to those in Fig. 3. Gene structure analysis showed that JmCOL proteins of the same subgroup usually shared similar exons and introns. However, the number of exons in a subgroup also varies.

Conserved motif prediction of JmCOL proteins using the MEME online website yielded 10 conserved motifs (Fig. 4B), and the motif types, numbers, and positions of the same branch genes were more consistent. The conserved motifs of COL proteins were further analyzed using the MEME software, and a total of 10 conserved motifs were identified and designated as motifs 1–10 (Fig. 4C). All genes contained motif 1 (CCT structural domain) and motif 2 (B-box structural domain). Furthermore, similar to evolutionary trees and gene structures, proteins with the same conserved structural domains tend to belong to the same subgroup.

JmCOLs promoters involved a large number of light responsiveness elements

In order to analyze the functional differences among the various members of the J. mandshurica JmCOL gene family, the promoters of the family genes were analyzed for cis-acting elements (Table 2). The results indicated that the walnut JmCOLs family genes possess eight light-response-related cis-elements, seven hormone-related cis-elements, five stress-related cis-elements, and cis-elements responsive to other growth and developmental processes. Among the 11 JmCOLs genes that contained BOX-4, a cis-acting element involved in light response, eight genes also contained ARE, a cis-acting regulator necessary for anaerobic induction. Additionally, eight genes contained CGTCA-motif, a cis-acting regulator involved in the response to MeJA. These findings suggest that the JmCOLs may be responsive to light, anaerobic induction, and MeJA. Furthermore, the prevalence of light-responsive related cis-elements indicates that the JmCOL family contributes significantly to light response and, to a lesser extent, potentially to hormone-related physiological processes.

Comparative genome collinearity analyses of COL genes between Juglans mandshurica and Arabidopsis thaliana

The distribution of the 15 JmCOL genes on the J. mandshurica chromosomes was analyzed based on the genomic information available for this species. As illustrated in Fig. 5A, gene duplication events were identified for six of these gene pairs, all of which were segmental duplications. To investigate the evolutionary relationship of COL genes, a covariance analysis was conducted on both J. mandshurica and A. thaliana. The results demonstrated that a total of 18 pairs of genes exhibited homology between the J. mandshurica and A. thaliana (Fig. 5B).

JmCOLs showed a tissue-specific expression in different tissues

In order to explore the expression pattern of JmCOLs genes in different tissues of J. mandshurica, we first studied the expression patterns of JmCOL family members in different tissues such as stems, fruits, leaves and flower buds by qRT-PCR. The effective experimental results of nine genes were obtained (Fig. 6), and these genes showed several different expression patterns. JmCOL 2, JmCOL4a, JmCOL 6 and JmCOL13 were mainly expressed in leaves. The transcription levels of JmCOL2 and JmCOL6 in leaves were at least two times that of other tissues. JmCOL16 a was mainly expressed in leaves, and the expression level was 14–23 times that of other tissues. JmCOL5 and JmCOL10 were abundantly expressed in fruits and flower buds, while JmCOL6, JmCOL13, JmCOL14 and JmCOL16 a were lowly expressed in flower buds. JmCOL4a was highly expressed in flower buds, fruits and leaves. Among the expression of JmCOL9a in different tissues, the highest expression level in stems was three times that in leaves, and this expression pattern was quite different from other members. These results indicate that the JmCOLs family may play an important role in all aspects of the growth and development of J. mandshurica, and several members with similar expression patterns may have similar functions.

Expression profiling of JmCOLs from transcriptome data

In the 12 physiological differentiation stage flower bud samples of J. mandshurica transcriptome, the gene expression coverage of each sample was shown in the form of density map. The FPKM density distribution map (Fig. S1) provides an overview of all gene expression patterns in the sample. In general, moderately expressed genes account for the majority, and a small number of genes are expressed at low and high levels. The FPKM value is proportional to the amount of gene expression. It can be seen from the graph that most of the 12 groups of samples overlap with each other. As shown in the attached figure, most of the intervals of the 12 groups of samples overlap with each other, indicating that the expression levels of the genes before and after treatment are similar, and only a very small part is different. The non-overlapping part may be the change of gene expression level caused by heterologous mechanism. In a heat map of FPKM values plotted for the JmCOLs genes, it can be seen that most members of the JmCOLs gene family are expressed in both male and female buds at the time of physiological differentiation, but the high expression of JmCOL5, JmCOL4a, JmCOL9b, and JmCOL11 in the type A samples implies that the female-preferred buds have a higher specificity of expression.

In the heat map of FPKM (Fig. 7A) values plotted for JmCOLs genes, it was found that most of the members of the JmCOLs gene family were expressed in both male and female flower buds during the physiological differentiation period, but the specific high expression of JmCOL5, JmCOL4a, JmCOL 9b, and JmCOL 11 in the type A samples revealed their possible involvement in protogynous bud formation and development.

At the physiological differentiation stage, correlation analysis based on the FPKM values of the transcriptome data was carried out with SPSS 26 and heat mapping was performed with TBtools. From the correlation analysis heatmap (Figs. 7B–7D), there were a large number of significant positive correlations in the expression of the JmCOLs gene family, (Fig. 7B). Notably there were also significant negative correlations between JmCOL4b/JmCOL9b in protandrous type of flower buds (Fig. 7C) and JmCOL9b/15 in protogynous type flower buds (Fig. 7B), and between the two types of flower buds, JmCOL5/16a, and JmCOL9b/14/15 in both flower buds (Fig. 7D). The specificity and correlations in the expression of JmCOLs reveal the potential involvement of members of this family in the process of bud sex differentiation during the early stages of bud differentiation.

Different types of flower buds and developmental periods have significant effects on the expression of JmCOL4a/5

According to the results of the heatmap, JmCOL4a/5 embodied specific expression in protogynous flower buds during the early stages of bud differentiation, and there were some positive and negative correlations with other genes. In order to see whether the expression of these two genes throughout bud differentiation is significantly differentiated among different types of buds, and thus to further validate the potential participation of the JmCOL gene family in the bud differentiation of huckleberry, we selected bud samples from ten developmental periods: April 2 (4.2), April 10 (4.10), April 14 (4.14), April 24 (4.24), April 30 (4.30), May 4 (5.4), May 13 (5.13), May 17 (5.17), May 20 (5.20), and May 22 (5.22) and performed qRT-Probe assays for the JmCOL4a/5 levels of JmCOL4a/5 were assayed by qRT-PCR. Statistics were plotted in Excel by one-way and multifactorial ANOVA in SPSS26 for s four types of flower buds (CC, CX, XC, XX) at different time periods.

Based on the results of statistical analysis, it was known (Table S5) that the expression of JmCOL4a/5 was significantly affected by the type of flower bud and the period of flower bud development (p < 0.05). JmCOL4a was lowly expressed during the physiological differentiation phase stage (April 2 to April 30), whereas it showed a trend higher than the physiological differentiation phase during the morphological differentiation phase (May 4-May 22) (Fig. 8). In terms of expression at different periods (Table S5), the expression of JmCOL4a in all four types of flower buds reached its highest value on May 20, with the expression being approximately 50–100-fold higher than that on April 14, the period with the lowest expression, respectively. From the viewpoint of flower bud types, JmCOL4a expression was overall higher in male than in female flower buds (Fig. 8). From the perspective of bud categorization of protogynous and protandrous, JmCOL4a expression was much higher in protogynous male buds (CX) than protogynous female buds (CC), and the difference in expression fluctuated between 6-10-fold at different times (p < 0.05). The expression of JmCOL4a in protandrous buds showed the same trend in male and female buds, but there was a difference in expression between protandrous male buds (XX) and protandrous female buds (XC), and the difference was mainly reflected on April 10, May 4, May 13 and May 17. Overall, the expression in protandrous (XX) buds was higher than that of protandrous female buds (XC).

In contrast to the expression pattern of JmCOL4a, at the stage of physiological differentiation (April 2 to April 30) JmCOL5 in the protogynous male flower buds (CX) appeared to have the highest expression of the four flower buds in this stage, which is in complete agreement with the results of the transcriptome data we measured at the beginning of the physiological differentiation (Table S5). In contrast, by the stage of morphological differentiation (May 4–May 22), JmCOL5 expression was higher in protandrous than in protogynous (Fig. 9), and was 2–4 times higher than that in protogynous. The highest value of JmCOL5 expression in protandrous male flower buds (XX) at different periods was about 30 times higher than the lowest value (p < 0.05). JmCOL5 had different expression patterns in protogynous and protandrous flower buds, and this difference was reflected in protogynous male flower buds (CX). In conclusion, JmCOL5 expression in these four types of flower buds was highest at the stage of morphological differentiation, except for the female-prior type male flower buds (CX), where the highest value was reached at the stage of physiological differentiation.