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Analysis of the molecular mechanism endogenous hormone regulating axillary bud development in Pinus yunnanensis

BMC Plant Biology volume 24, Article number: 1219 (2024) Cite this article

Abstract

Background

P. yunnanensis, a distinctive economic tree species native to Yunnan Province in China, possesses axillary buds that serve as superior material for asexual propagation. However, under natural growth conditions, the differentiation of these axillary buds is notably scarce. In this study, we employed decapitation to stimulate the development of axillary buds in P. yunnanensis. Subsequently, we assessed the phytohormone levels in both axillary and apical buds, and conducted a comprehensive transcriptomic analysis complemented by RT-qPCR validation.

Results

We found that decapitation could effectively promote the releases of the axillary buds in P. yunnanensis. The levels of cytokinin, auxin, gibberellin and abscisic acid in axillary buds were higher than those in apical buds, and the difference in gibberellin levels was the greatest. The transcriptome sequencing results were highly reproducible, and the relative expression levels of the 13 genes screened were highly consistent with the FPKM value trend of transcriptome sequencing. There were 2877 differentially expressed genes (DEGs) between axillary buds and terminal buds, and 18 candidate genes (CGs) involved in axillary bud release were screened out. A total of 1171 DEGs were identified during the analysis of axillary bud growth, and 14 CGs involved in axillary bud growth and development were screened out. GO and KEGG enrichment analysis were performed on the DEGs. Furthermore, combined with the results and discussion, the functions of the candidate genes were analyzed and a possible regulatory network was constructed.

Conclusion

The findings and discussions indicated that the development of axillary buds in P. yunnanensis is predominantly governed by cytokinin, gibberellin, strigolactone, and auxin, as well as their biosynthesis and regulatory genes, which are crucial to the development of these buds. This study has, to some extent, bridged the research gap concerning the development of axillary buds in P. yunnanensis and has provided foundational data to support further research into the developmental mechanisms of these buds and the establishment of asexual propagation cutting nurseries.

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Background

P. yunnanensis, a species within the Pinus L. genus, is predominantly found in southwestern China [1], Within Yunnan Province, it stands out as a distinctive economic forest tree species, offering significant industrial, ecological, and economic benefits [2, 3]. The quality of the axillary buds in P. yunnanensis is crucial for its morphology and industrial applications. Nevertheless, current research on P. yunnanensis primarily focuses on the types and functions of secondary metabolites [4, 5], population genetic diversity [6,7,8], stand composition structure [9], as well as pests, diseases, and their management strategies [10, 11], among other areas.

The quality of axillary buds in P. yunnanensis plays a crucial role in shaping its morphology and is vital for industrial applications. Decapitation emerges as a pivotal technique to stimulate the germination of these buds within the plant kingdom [12, 13]. Following decapitation, the concentrations of endogenous plant hormones undergo significant alterations, thereby influencing the development of axillary buds [14]. Upon detecting hormonal signals, plants react by activating and fostering the growth of axillary buds [15]. Initial investigations have highlighted that auxin is pivotal in sustaining apical dominance. Decapitation results in a reduction of IAA (Indole-3-acetic acid) levels within the plant’s stems, and the overexpression or disruption of genes responsible for IAA synthesis can lead to notable changes in auxin concentrations [16]. Further exploration into the functional impacts of endogenous plant hormones has revealed that cytokinins, gibberellins, and strigolactones all contribute to the growth and development of axillary buds [17]. Cytokinins and gibberellins exert a positive regulatory effect [18], whereas auxins and strigolactones exert a negative influence on the development of axillary buds [19]. Nevertheless, the levels of plant endogenous hormones are governed by key regulatory, synthetic, or degradative proteins [20, 21].

Cytokinin biosynthesis genes exhibited upregulation, whereas the expression of CKX (cytokinin oxidase) genes was suppressed, leading to heightened cytokinin levels and the activation of dormant axillary buds [22]. Cytokinins possess the capacity to stimulate WUSCHEL (WUS) expression, thereby facilitating the initiation of plant axillary meristems [23, 24]. The expression levels of genes involved in gibberellin synthesis, oxidation, and signal transduction experienced significant alterations both before and after decapitation [25]. Decapitation triggered the up-regulation of gibberellin biosynthesis genes. Gibberellin and abscisic acid played roles in modulating the transcription and translation of embryo-related genes through signal transduction pathways [26]. The overexpression of the ABF gene resulted in an increase in abscisic acid content within runners [27]. Strigolactones inhibited the development of axillary buds in plants [28], and mutations in their synthetic genes, ccd7 and ccd8, promoted the release of axillary buds [29].

The reasons for the loss of apical dominance in dwarf P. yunnanensis were analyzed through transcriptome and metabolome, pointing out that GA 2-oxidase (GA2ox) may regulate the development of lateral branches. It was determined that the contents of gibberellins and abscisic acid in dwarf P. yunnanensis were high [30], and dwarf P. yunnanensis showed dominant characteristics under drought stress conditions [31]. In the study of P. yunnanensis seedlings, it was found that there were significant differences in the levels of endogenous hormones in P. yunnanensis at different classification levels, and it was shown that hormone signal transduction and regulatory genes regulate the growth and development of P. yunnanensis [32].

In summary, the growth and development of axillary buds are governed by endogenous hormone levels, and transcriptomics serves as a crucial tool for uncovering the molecular mechanisms underlying plant growth and development. To elucidate the developmental mechanisms of axillary buds in P. yunnanensis, our study primarily employed transcriptomic analysis to compare axillary buds with terminal buds of P. yunnanensis, and to investigate the differential gene expression in axillary buds across various growth conditions. The goal is to furnish substantial data to support research into the growth and development of axillary buds in P. yunnanensis, and to identify candidate genes that modulate hormones associated with axillary bud development.

Results

Decapitation stimulates the release and growth of axillary buds

The axillary bud differentiation potential of P. yunnanensis seedlings was initially weak; however, decapitation notably enhanced this capacity. Thirty days post-decapitation, a significant increase in the number of axillary buds was observed (Fig. 1-A and B). Hormone level assessments of both axillary and apical buds (Fig. 1-C) revealed that the concentrations of ZT, GA, IAA, and ABA in axillary buds surpassed those in apical buds, suggesting that the sprouting of P. yunnanensis axillary buds is governed by endogenous plant hormones. Apart from ZT, the levels of other endogenous hormones exhibited marked differences, with GA3 showing the most significant variance, implying that GA3 might predominantly influence the growth of released axillary buds. The axillary buds of P. yunnanensis experienced rapid growth from April to July (Fig. 1-D), peaking in May and June, with growth rates starting to diminish from June to July. This indicates that the growth rate of P. yunnanensis axillary buds was initially swift and subsequently slowed from April to July (Fig. 1-E).

Fig. 1
figure 1

Decapitation stimulates the growth and development of axillary buds in P. yunnanensis. (A; B) The growth status and average number of axillary buds in P. yunnanensis 30 days post-decapitation; (C) Variations in endogenous hormone levels between axillary and terminal buds, with ZT representing Zentin, GA indicating Gibberellic acid, IAA denoting Indole-3-acetic acid, and ABA referring to Abscisic acid; (D) The growth progression of P. yunnanensis from April to July following decapitation; (E) The development of axillary buds in P. yunnanensis across various time intervals. Scale: 1 cm

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Transcriptome sequencing outcomes

Throughout the sequencing process, sample quality was stringently monitored and met the established sequencing criteria (refer to Appendix Tables 1, 2, 3 and 4). The sequencing outcomes indicated that the raw reads throughput for each sample varied between 38,610,298 and 51,625,016 (refer to Fig. 2-A and Appendix Table 1–1). Following data filtering, the percentage of clean reads surpassed 99.8% (Fig. 2-B). Notably, the Q20 proportion for each sample exceeded 96.0%, while the Q30 proportion exceeded 90.0%. The GC content ranged from 46.55 to 48.01% (refer to Appendix Table 1, and 2), and the base composition of each sample was essentially balanced. The high-quality clean reads were aligned with the publicly accessible Pinus taeda genome database, resulting in a mapped reads proportion of 78.08-81.57%, and a unique mapped reads proportion of 75.42-78.37% (refer to Appendix Tables 1, 2 and 3). Beyond the overall alignment with the genome, the sequenced reads were further aligned with the exon regions of genes. The alignment ratio for all treatments studied exceeded 56.0%, with the lowest ratio observed in the intron region, which was below 8.5%. The alignment ratio with the intergenic region fluctuated between 32.6% and 34.8% (Fig. 2-C). Based on the FPKM value, the expression distribution diagram revealed a high level of consistency in gene or transcript expression across all samples (Fig. 1-D). Principal Component Analysis (PCA) (Fig. 1-E) demonstrated that the clustering results among samples were robust, suggesting that the sequencing data possessed excellent repeatability. In conclusion, the transcriptome sequencing results were robust, offering quality assurance for subsequent analyses.

Fig. 2
figure 2

Transcriptome sequencing results. (A) Data preprocessing value distribution diagram; (B) Data preprocessing percentage distribution diagram; (C) Comparison reference area statistics diagram; (D) Violin plot of gene expression; (E) Sample principal component analysis

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Analysis of DEGs between axillary and apical buds of P. yunnanensis

The differential gene analysis yielded results indicating that 2,877 genes exhibited significant changes in expression (Fig. 3-A; B), with 1,759 up-regulated and 1,118 down-regulated, a trend showing a higher count of up-regulated genes (Appendix Table 2). Cluster analysis of these differentially expressed genes revealed two primary categories (Fig. 3-C). Further, Gene Ontology (GO) enrichment analysis (Fig. 3-D) identified that these genes were classified into three main ontologies: biological process, cellular component, and molecular function. Within the top 20 enriched GO pathways, 17 were categorized under biological processes, 1 under cellular components, and 2 under molecular functions. Specifically, the pathway with the highest number of significantly enriched genes within the biological process was the response to stimulus (GO: 0050896), followed by response to chemical (GO: 0042221). The prevalence of up-regulated genes compared to down-regulated genes indicates that decapitation induces alterations in the plant’s internal milieu, leading to the activation of genes responsive to stimuli and affecting the development of axillary buds in P. yunnanensis.

KEGG enrichment analysis (Fig. 3-E) revealed that among the top 20 enriched KEGG pathway categories, the pathway with the highest number of significantly enriched genes was Metabolism, succeeded by Environmental Information Processing. Within the Metabolism category, the pathway with the greatest number of enrichments was Metabolic pathways (ko01100), followed by Biosynthesis of secondary metabolites (ko01110). Notably, within the Organic Systems category, the Plant hormone signal transduction (ko04075) pathway was identified, which included a total of 38 differentially expressed genes, comprising 26 up-regulated and 12 down-regulated genes.

Fig. 3
figure 3

Analysis of Differentially Expressed Genes and Top Pathways. (A) Statistics of gene number differences; (B) Volcano plot illustrating differences, with values represented as log2(FC); (C) Heatmaps of different comparative analyses; (D) GO enrichment circle diagram, featuring the top 20 GO terms with the lowest Q-values; (E) KEGG enrichment circle diagram. The first circle highlights the top 20 pathways, with the number of genes indicated by the outer circle’s scale. Different colors correspond to distinct clusters; the second circle displays the quantity and Q-values of pathways within the background gene set, where a greater number of genes is represented by a longer bar, and the Q-value is indicated by color intensity, with more significant values shown in red; the third circle illustrates the gene ratio percentages, with dark purple denoting the gene ratio percentage and light purple indicating the gene ratio circle; the fourth circle represents the RichFactor values for each pathway, with the background grid lines indicating increments of 0.1

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Sequencing results verification and identification of key genes involved in axillary bud development

In this study, 13 genes (refer to Appendix Table 3 for the primer list) were utilized to validate the sequencing outcomes through qPCR analysis (Fig. 4-A). It was observed that the relative expression patterns of these selected genes were highly consistent with the trends seen in the transcriptome sequencing data, thus affirming the reliability of the transcriptome sequencing results. By examining genes associated with plant hormone synthesis and regulation, a total of 18 genes potentially implicated in axillary bud differentiation were identified (Fig. 4-B). Among the auxin-related genes, the expression of auxin-binding protein genes ABP19 and ABP20 was notably downregulated, whereas the auxin synthesis gene SAUR50 exhibited significant upregulation. Within the cytokinin gene family, the cytokinin oxidase gene CKX5 showed a marked decrease in expression. For gibberellin biosynthesis genes, the gibberellin oxidase genes GA2ox2, GA2ox8, GA2ox9, and the gibberellin receptor gene GID1C were all significantly downregulated. No significant differential expression was detected in strigolactone biosynthesis genes, but its receptor gene DAD2 was notably upregulated. Abscisic acid, a critical hormone in abiotic stress responses, had its synthetic gene NCED3 significantly upregulated in the axillary buds of decapitated plants. In conclusion, genes involved in hormone synthesis and signal transduction pathways exhibit significant differential expression in axillary buds, encompassing auxin, cytokinin, gibberellin, strigolactone, and abscisic acid. Hence, the development of axillary buds in P. yunnanensis is modulated by these aforementioned hormones.

Fig. 4
figure 4

Analysis of Candidate Genes for Axillary Bud Development in P. yunnanensis. (A) Confirmation of Transcriptome Data Outcomes; (B) Heatmap Depicting the Expression Levels of Key Candidate Genes Involved in Axillary Bud Development in P. yunnanensis, Represented as log10(FPKM)

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Analysis of genes regulating growth of axillary buds in P. yunnanensis

After analyzing the growth dynamics of axillary buds of P. yunnanensis, transcriptome analysis was performed on axillary buds of P. yunnanensis from April to July. Among the combinations (Fig. 5-A), the one with the most differentially expressed genes was Ax1-vs-Ax4, with a total of 4,680 genes, while the one with the least differentially expressed genes was Ax1-vs-Ax3, with a total of 1,892 genes (Appendix Tables 4, 5 and 6). There were 1,171 differentially expressed genes shared by the combinations (Fig. 5-B). Functional enrichment analysis was performed on the differentially expressed genes of the three combinations. Among the top 20 enrichment results, GO was mainly enriched in biological processes (Fig. 5-C; Appendix Fig. 1), and KEGG was mainly enriched in metabolism (Fig. 5-D; Appendix Fig. 1).Through the screening and analysis of differentially expressed genes related to hormones, it was pointed out that the genes involved in the growth and development of axillary buds are mainly the synthesis or regulation of auxin, gibberellin, cytokinin, and strigolactone. Abscisic acid biosynthesis genes NCED3, NAC2, and JA2 were significantly down-regulated, and the role of abscisic acid is to participate in abiotic stress, so the growth regulation effect of abscisic acid on axillary buds may be small. The candidate genes screened are indicated in Fig. 5-E.

Fig. 5
figure 5

Analysis of axillary bud growth and development in P. yunnanensis. (A) Statistics of differentially expressed genes across groups; (B) Venn diagram illustrating differentially expressed genes between groups; (C-D) GO and KEGG enrichment circle maps for the top 20 differentially expressed genes in “Ax1-vs-Ax2”; (E) Heat map displaying candidate genes involved in regulating axillary bud growth, with values represented as log2(FPKM)

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Discussion

Effects of decapitation on P. yunnanensis

The levels of plant hormones in P. yunnanensis were also influenced by decapitation, which causes irreversible damage to the seedlings. Consequently, it was crucial to comprehend the mechanism through which endogenous hormones in P. yunnanensis stimulate the development of axillary buds. The primary site of auxin synthesis was the shoot apex or root apex. Following decapitation, auxin was rapidly depleted, leading to a significant drop in IAA levels over a brief period. During the development of axillary buds in P. yunnanensis, both auxin levels and synthetic genes were upregulated, suggesting that auxin in these buds facilitates their growth. Elevated levels of gibberellins and cytokinins have been detected in the axillary buds of decapitated P. yunnanensis, aligning with findings that the application of cytokinins and gibberellins promotes axillary bud development in plants. Decapitation induced sucrose accumulation in plants, and sucrose, in turn, stimulates cytokinin synthesis and suppresses strigolactone synthesis, thereby fostering the growth and development of axillary buds. In P. yunnanensis, the downregulation of CKX5 gene expression and the high cytokinin levels in axillary buds indicate that cytokinin plays a role in promoting their development. ABA was involved in plant abiotic stress responses, and its content in the axillary buds of P. yunnanensis increases notably after decapitation, with the synthesis gene NCED3 being significantly upregulated. However, as the axillary buds of P. yunnanensis mature, NCED3 and other genes that regulate ABA synthesis were downregulated. This suggests that decapitation triggers ABA synthesis, which was primarily associated with the abiotic stress response in P. yunnanensis.

Analysis of the regulatory mechanism of axillary bud development in P. yunnanensis

The developmental state of axillary buds was a crucial determinant of plant architecture, influenced by endogenous hormones, environmental factors, and key genetic components [33]. The TCP gene family member TB1/BRC1 was recognized as a pivotal inhibitor of lateral bud differentiation [34,35,36]. Strigolactones suppress axillary bud differentiation by enhancing BRC1 expression [19, 37, 38], and mutations in the strigolactone biosynthesis genes CCD7 and CCD8 lead to plants with multiple branches [39, 40]. DAD2/D14 was a strigolactone-responsive protein that facilitates the breakdown of strigolactones [41, 42], and plants with mutations in DAD2/D14 exhibit insensitivity to strigolactone analogs and display multiple branching or tillering traits [43,44,45]. The DAD2 gene was highly expressed in axillary buds [44], and our research indicates that DAD2 gene expression remains elevated during axillary bud growth, suggesting that DAD2 may contribute to the promotion of axillary bud development. There was an interplay between cytokinin and strigolactone regulatory pathways [46, 47]. In plants with impaired strigolactone synthesis, cytokinin levels were maintained at elevated levels [48], and strigolactones decrease cytokinin levels by inducing CKX gene expression. Cytokinins, in turn, regulate the upregulation of DWARF53, a negative regulator of strigolactone biosynthesis, thereby inhibiting the production of strigolactones [49]. We did not proceed with further analysis in this instance. Naturally, our future efforts will concentrate on investigating the role of strigolactones in the development of axillary buds in P. yunnanensis.

Cytokinins actively contribute to the release of axillary buds. These buds emerge from the axillary meristem, and their fate was dictated by the WUS and STM genes [50, 51]. Cytokinins, however, orchestrate the upregulation of WUS gene expression, which in turn fosters the differentiation of axillary bud meristems [24]. WUS emerges as a key player by breaking bud dormancy through the recruitment of TPL to suppress TCP12 expression [52]. Consequently, cytokinins negatively regulate BRC1 expression, thereby facilitating the release of axillary buds. In the case of P. yunnanensis, the expression of the CKX5 gene in axillary buds was downregulated relative to that in apical buds, ensuring a high level of cytokinin throughout the development of axillary buds, which in turn promotes their growth.

Gibberellin was crucial for the development of buds. The application of exogenous gibberellins can markedly improve the differentiation and growth of axillary buds, and they negatively regulated TB1 to stimulate rice tillering [53]. Our research findings indicate that the gibberellin concentration in axillary buds was considerably higher than in apical buds, and the gibberellin oxidase genes GA2ox2, GA2ox8, GA2ox9 were notably down-regulated in axillary buds [54,55,56,57], a phenomenon that is pivotal in maintaining a high gibberellin level within these buds. The gibberellin receptor protein GID1 can bind with DELLA proteins to modulate gibberellin biosynthesis [58,59,60], and in rice, DELLA proteins interact with the tiller number regulator MONOCULM 1 (MOC1) to collaboratively control plant height and tillering balance [61]. However, the expression of GID1C was significantly elevated in axillary buds, suggesting its significant role in the development of these buds.

Fig. 6
figure 6

Hormone-regulated axillary bud development after decapitation of P. yunnanensis

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Auxin played an indirect regulatory role in axillary bud development and does not directly act on genes that control axillary bud differentiation. During axillary bud development, it enhanced the resistance of axillary bud release by promoting the synthesis of strigolactones and inhibiting the synthesis of cytokinins [62]. In plants, auxin synthesized by the apical buds did not directly act on the axillary buds, but inhibited the efflux of auxin in the axillary buds, thereby inhibiting the development of axillary buds [33]. However, auxin in axillary buds induces gibberellin synthesis during axillary bud growth and promotes axillary bud growth [17, 33]. Therefore, during the rapid development stage of axillary buds of P. yunnanensis, high expression levels of auxin synthesis genes such as IAA9 and IAA27 were detected. Therefore, the reason for the high auxin level in the axillary buds after release was explained. In summary, auxin, cytokinin, gibberellin, and strigolactone interact with each other to regulate the development of axillary buds in P. yunnanensis, and their biosynthesis-related genes regulate hormone levels to control the development of axillary buds. A simple map of endogenous hormones regulating axillary bud development in P. yunnanensis was designed (Fig. 6).However, the regulatory network of axillary bud development is complex, and whether hormone-regulated genes directly act on axillary bud development remains to be further studied.

Conclusion

This investigation revealed that decapitation can alter the internal hormonal levels in P. yunnanensis, thereby effectively stimulating the release of axillary buds. The precise transcriptional profiles of axillary and apical buds, along with the developmental process of axillary buds, have identified candidate genes involved in hormonal regulation during axillary bud development in P. yunnanensis. This offers significant data support for the exploration of hormonal regulation in the axillary bud development network. Furthermore, the regulatory impacts of hormones such as auxin, cytokinin, gibberellin, and strigolactone, as well as their synthetic genes, on the development of axillary buds in P. yunnanensis were examined. Notably, gibberellin plays a crucial role in the development of axillary buds. This study has comprehensively analyzed the hormones, along with their synthesis and response genes, that govern the emergence and growth of axillary buds in P. yunnanensis. It has also identified several candidate genes, providing valuable data support for future research into the branching of P. yunnanensis.

Materials and methods

Plant materials

Seeds of P. yunnanensis were harvested from the Midu P. yunnanensis clone seed garden (Yun S-CSO-PY-001-2016). These seeds were subsequently sown into plug trays, and after three months, seedlings exhibiting similar growth conditions were chosen for transplantation into seedling pots (with a bottom diameter of 16 cm, a diameter of 24 cm, and a height of 20 cm). Seedling maintenance primarily consisted of irrigation and weeding practices. It was recommended to water the plants every 3–5 days, ensuring thorough saturation. The experimental site was established within the greenhouse facilities of Yunnan Jicheng Garden Technology Co., Ltd., situated in Mile City, Yunnan Province, China. This city is situated within a subtropical monsoon climate zone, boasting an average annual temperature of 17.1 ℃, an average annual precipitation of 950.2 mm, a relative humidity of 73%, an average annual sunshine duration of 2131.4 h, and a frost-free period spanning 323 days.

Sample handling and sampling

The previous research of the research group showed that the period from March to July was the period of rapid growth of P. yunnanensis. The axillary bud differentiation ability of P. yunnanensis seedlings was weak.Therefore, the samples were decapitated in March (with a stem height of 10 cm) and marked as “Dec”, with a total of 20 plants. The samples that were not decapitated were marked as “Col”, with a total of 20 plants.

Decapitation will damage the plants, so the required samples were collected after they had grown for one month. Sampling was carried out from April to July, once a month, at the beginning of each month, and three replicates were set for each sample.The apical bud was marked as “Ap”; the axillary bud was marked as “Ax” (the axillary bud samples from April to July are marked as “Ax1; Ax2; Ax3; Ax4”). When sampling, quickly took samples on ice and put them into a centrifuge tube placed in liquid nitrogen. After sampling, stored the samples in a -80℃ refrigerator. Each sample was from 5 P. yunnanensis trees and replicated 3 times.

Growth indicators and plant hormone determination

When counting the number of axillary buds, the length of the axillary buds must be more than 0.5 cm. The growth length of the axillary buds refers to the length of the axillary buds that grow within a period of time. When measuring, select the three axillary buds with the best growth status for each plant. Hormone determination was commissioned to Suzhou Keming Biotechnology Co., Ltd., which used high performance liquid chromatography (RIGOL L3000) to determine the hormone content. The hormones determined included IAA, ZT, GA3, and ABA.The above data were sorted using Excel software, and variance analysis was performed using IBM SPSS Statistics 26 statistical analysis software.

Transcriptome sequencing analysis

The transcription group sequencing (Transcriptome sequencing with references, the reference genome specie was Pinus taeda, https://ftp://plantgenie.org/data/conie/) entrusting Guangzhou Kidio Biotechnology Co., Ltd. A total of 5 samples were sequensed. Mainly conduct sequencing library construction, data quality control [63], sequence comparison and evaluation [64], gene expression calculation [65], correlation analysis.

Screening and analysis of DEGs

The DESeq algorithm [66] was used to obtain the FDR value (False Discovery Rate), and genes with FDR < 0.05 and |log2FC| > 1 were identified as DEGs. The software BLAST and KOBAS were used to perform functional annotation of DEGs, including GO annotation, functional classification and KEGG annotation. GO enrichment analysis of DEGs was performed using topGO [67] software, and KOBAS software was used to perform KEGG functional enrichment analysis of DEGs [68].

Verification of sequencing results

In order to verify the reliability of transcriptome (RNA-seq) data, real-time fluorescence quantitative PCR (qRT-PCR) was performed on the transcriptome data. The extracted RNA was reverse transcribed using the reverse transcription kit (R223) of Novazonics Biotech Co., Ltd., and the RNA was reverse transcribed into cDNA and stored at -80 °C for future use. The internal reference gene was PITAhm_003261 [32](Obtained through preliminary screening by the research team.), and 13 genes were selected for the experiment(Including cytokinin, abscisic acid, auxin, gibberellin.). The instrument used was the Roche Light Cycler® 480 II System. qPCR reaction conditions: denaturation at 95 °C for 90 s; 95 °C, 5 s, 40 cycles; 60 °C, 15 s; 72 °C, 20 s. Each sample was replicated three times; the relative expression of genes was calculated using the 2-ΔΔCt method [69].

Screening of candidate genes related to axillary bud development

The differential gene data of the “Ap-vs-Ax1” combination were selected as the database for screening candidate genes related to the regulation of axillary bud release in P. yunnanensis. The “Ax1-vs-Ax2; Ax1-vs-Ax3; Ax1-vs-Ax4” differential gene database was selected as the database of relevant candidate genes regulating the growth of axillary buds of P. yunnanensis. During the screening process, the main focus is on genes that synthesize or regulate endogenous plant hormones such as auxin, gibberellin, cytokinin, abscisic acid, and strigolactone.

Data availability

The raw transcriptome data have been uploaded to NCBI (Accession Number: PRJNA1161604). The gene expression data and CDS sequence files have been collated and placed in the supplementary files.

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Acknowledgements

Here, I would like to express my sincere gratitude to my supervisors YL X (Professor) and NH C (Associate Professor) for their guidance in my experimental work and article writing.Secondly, I would like to thank my research group partners, and We are thank Guangzhou Genedenovo Biotechnology Co., Ltd for assisting in sequencing.

Funding

This research was supported by the Key Project of Joint Funds of the Basic Agricultural Research of Yunnan Province (grant number 202301BD070001-152), and Joint Funds of the Basic Agricultural Research of Yunnan Province (202301BD070001-035), National Natural Science Foundation of China (32360381), Yunnan Province Ten Thousand People Plan Youth Top Talent Project Funding (YNWR-QNBJ-2019-075).

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Authors and Affiliations

  1. Key Laboratory of Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University, Kunming, Yunnan Province, China

    Haihao He, Junfei Xu, Nianhui Cai & Yulan Xu

  2. Laboratory of National Forestry and Grassland Administration on Biodiversity Conservation in Southwest China, Southwest Forestry University, Kunming, Yunnan Province, China

    Haihao He, Junfei Xu, Nianhui Cai & Yulan Xu

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  1. Haihao He
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  2. Junfei Xu
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  4. Yulan Xu
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Contributions

HH H: Experimental design, data analysis, Writing-original draf.JF X: Sample collection. NH C & YL X: Project management, article modification.

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Correspondence to Nianhui Cai or Yulan Xu.

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He, H., Xu, J., Cai, N. et al. Analysis of the molecular mechanism endogenous hormone regulating axillary bud development in Pinus yunnanensis. BMC Plant Biol 24, 1219 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12870-024-05819-6

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BMC Plant Biology

ISSN: 1471-2229

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