Ecological and economic value may be found in the monoecious plant Pinus koraiensis, an evergreen conifer of Pinaceae with a tree height of 40 m and a breast height diameter of 1 m (Li et al., 2022; Park et al., 2016; Ren et al., 2022). P. koraiensis is mostly distributed in China’s Changbai Mountains, Laoyeling Mountains, Zhangguangcailing Mountains, Xiaoxinganling Mountains and Wanda Mountains, as well as in Japan, the Far East region of Russia and the Korean Peninsula (Aizawa et al., 2012; Shi et al., 2020; Wei et al., 2022; Zhang et al., 2022). It has state approval to be designated as a secondary wild plant for national protection (Li et al., 2020). Due to its plentiful lipid, amino acids, proteins, and other nutrients that can successfully decrease blood lipids and cholesterol in humans, P. koraiensis seed kernels have become one of the most significant nut foods accessible (Song et al., 2021; Ryuk et al., 2022; Wang et al., 2022). It frequently takes the natural P. koraiensis 20 to 30 years to produce cones due to its slow growth cycle. And its inconsistent blooming time and low yield during cone setting result in an exceedingly unpredictable seed production, which results in farmers suffering huge financial losses (Li et al., 2021; Sun et al., 2021; Wu et al., 2022; Wang et al., 2021a). Here, P. koraiensis seed yield is mostly influenced by the number and growth development of male and female cones. There has not been any information published about the morphologic change, hormone levels, or gene expression that occurs while P. koraiensis male and female cones develop. Therefore, it is crucial to investigate the growth developmental characteristics and mechanism of this species, as this knowledge may help to increase P. koraiensis seed production.

Plants go through several developmental stages during their life cycle, but the formation of flower is the most significant stage since it signifies the critical transition from vegetative to reproductive growth (Wellmer and Riechmann, 2010; Liu et al., 2016; Wang et al., 2021b). The development of flowers is regulated by both the external environment and their internal genes, which have long built a sophisticated and precise gigantic regulatory network (Mouradov et al., 2002; Ó’Maoiléidigh et al., 2014; Chen et al., 2018). Numerous elements, including photoperiod, temperature, phytohormones, vernalization, autonomy, and gene regulation, have been demonstrated to mediate the flowering process in plants with the advent of molecular biology and genetics (Cao et al., 2021). However, it has been suggested that phytohormones hold the key to controlling floral sex differentiation and development (Cheng and Zhao, 2007). The impact of exogenous hormones on Castanea henryi flowers development was discussed by Fan et al. The findings demonstrated that gibberellins and abscisic acid were mostly deposited in male flowers, while cytokinins were significantly more abundant in female flowers than in male flowers, demonstrating the dynamic changes in hormones throughout floral development (Fan et al., 2017). Transcriptome and hormone analysis of major buds in Crocus sativus L. revealed that GA₄ content was a prerequisite for flower induction and development (Renau-Morata et al., 2021). Additionally, research has demonstrated that jasmonate is essential for reproductive activities such as plant male fertility and sex differentiation (Yuan and Zhang, 2015), and which mediates plant resistance to against necrotrophy (Zheng et al., 2022). Moreover, through direct ABA biosynthesis promotion and callose synthetase gene CALLOSE SYNTHETASE 1 activation, the poplar SVL protein improves terminal bud endodormancy (Gao et al., 2021). Furthermore, many reports using angiosperms as model plants provide only limited references to gymnosperms. In the study of Pinus radiata, it was mentioned that the gibberellin complex (GA1, GA3, GA4, GA7, GA9) were closely related to the development of cone buds, which was mainly manifested in the significant differences in the content of different development stages (Ross et al., 1984). ABA has a complex regulatory mechanism for plant growth and development. When the ABA metabolic pathway is activated and the content decreases, the dormancy period of male cones of Pinus tabulaeformis is shortened and the growth of stems is accelerated (Zhang et al., 2022).

Genes have the ability to regulate the phenotypic characteristics of plants. Previous studies have identified the role and molecular mechanism of some key regulatory genes in plant flower growth. By triggering FT activity, CO controls the synthesis and transmission of flowering signals in the model plant Arabidopsis thaliana (An et al., 2004). FOREVER YOUNG FLOWER 1 (FYF1) and FYF2 are activators that, when combined, promote flower senescence and block flower abscission, respectively (Chen et al., 2022). The SOC1 gene also plays a key role in the flower induction phase of Persian walnut flower growth, and the FT gene is strongly expressed at the flowering stage (Hassankhah et al., 2020). In addition, the researchers also concluded that TFL2, CO, NF-YC1 and NF-YC4 have a positive regulatory role in the formation of cones by identifying the function of gene families in male cones of P. tabulaeformis (Guo, 2020). A set of transcription factors known as the MADS-box gene family particularly recognizes and binds to distinctive DNA sequences. It controls a variety of gymnosperms and angiosperms plant development processes, such as the growth of flowers/cones, fruits, leaves, and roots (Taylor et al., 2002; Koshimizu et al., 2018; Susanne et al., 2019; Niu et al., 2022). At the bud stage and flower development stage of the chickpea, one study found that a total of 18 MADS-box genes were discovered, and the expression differential was the most significant of all transcription factors (Singh et al., 2013). A. thaliana contains 109 MADS transcription factor family. According to research, MADS domain proteins are present at different phases of plant flower development, and other transcription factor families work in conjunction with the MADS-box gene family to support the formation and development of flower organs (Smaczniak et al., 2012). In conifers, MADS gene family are thought to be a regulatory mediator for the transition from juvenile to adult (Carlsbecker et al., 2004; Carlsbecker et al., 2013). For example, Ma et al. focused on the functional characteristics of the MADS gene family by performing time-dynamic transcriptome analysis on P. tabulaeformis. The results showed that it was not only closely related to aging, but also played a key role in the transition from vegetative growth to reproductive growth of P. tabulaeformis (Ma et al., 2021). Similar conclusions were also confirmed in Picea abies (Akhter et al., 2018).

In the field of conifers development biology, male and female cone development has long been a focus of investigation. Improved seed breeding, introduction, high yield, and steady cultivation can all be aided by understanding how plants make cones (Goslin et al., 2017; Zheng and Xia, 2022). High-performance liquid chromatography and transcriptome sequencing methods were employed in this study to examine the changes in hormone and gene levels at various stages of growth in P. koraiensis male and female cones. We started by performing morphological observations on the main growth developmental stage after the formation of male and female cones. Second, the amounts of eight plant hormones that are produced by the body were examined in cones at three significant growth phases. Finally, gene-specific alterations related to three significant developmental stages were examined using transcriptome sequencing. Through systematic phenological observation, hormone determination, and gene level analysis, this study seeks to better understand the relationship between male and female cones development and hormone content and genes. It also provides a useful reference for P. koraiensis genetic engineering breeding.

The primary plant reproductive organ is the flower/cone. Its primary biological role is to make seeds by fusing female egg cells with sperm cells (Hoenicka et al., 2016; Dai et al., 2018; Diao et al., 2022). The flowering process is a crucial part of a plant’s whole life cycle and is primarily influenced by both internal physiological and external environmental influences. Early fruiting, high yield, and plant stability are all strongly correlated with the timing, quantity, and quality of flower growth development (Cerdan and Chory, 2003; Blackmore et al., 2007; Yang et al., 2010). P. koraiensis has significant commercial and ecological importance as a lumber and medicinal plant. However, P. koraiensis has a lengthy cone development period, a significant proportion of male cone, and a small proportion of female cone, and is therefore prone to lower yield. Additionally, some female cones will undergo abortion, which will lead to unsteady seed setting. Understanding the P. koraiensis cone flowering phase and molecular mechanism is crucial for increasing yield and production applications. To fill in the gaps in this crucial developmental process and lay a strong foundation for morphology and genetics research of P. koraiensis, the main process of male and female cone growth development of P. koraiensis was observed in this study. The molecular regulatory mechanism during cone growth was analyzed by transcriptome sequencing technology.

Understanding the process of cone growth development can be a guide for managing P. koraiensis production and artificial pollination at the right moment. In this study, we discovered that at the earliest stages of growth development, cone buds and leaf buds could not be separated. At 120 DAY, leaf buds were still extending with the rise in temperature, while cone buds were steadily thickening and displaying features. Gymnosperms such as P. tabulaeformis (Niu et al., 2016), Pinus bungeana (Liu, 1985), and Pinus yunnanensis (Chen and Wang, 1982) are widely known for taking two to three years from cone buds to cones. Additionally, a number of studies have noted that taxus plants are fertilized in the second year after pollination (Carlsbecker et al., 2013; Niu et al., 2022). The pollination period of P. koraiensis in this study was found to be between 160 and 165 DAY. When pollination was successful, the male cones withered while the female continued to grow until the second year, when the cones reached maturity. The study of the particular fertilization process, however, has not been documented, and this will be a critical scientific issue that must be resolved in the future. Furthermore, the link between cone buds and leaf buds is unknown, and distinguishing coniferous bundle cone buds has always been a contentious issue.

In response to certain environmental cues, plant cells actively manufacture chemicals known as phytohormones that can control physiological responses in plants. Phytohormones have significant regulatory effects on plant cell division and elongation, tissue and organ differentiation, blooming and fruiting, maturation and senescence, and dormancy and germination (Cheng et al., 2002; Ashikari et al., 2005; Kurakawa et al., 2007). All phases of flower development involve the synthesis of GA, which is crucial for flower growth development (Plackett et al., 2011; Bai and Xu, 2013; Song et al., 2013). The GA content in this study was significantly altered at various phases (P<0.05), demonstrating that GA is essential for the growth of P. koraiensis male cone. Additionally, the growth development of the male cone showed that the MS3 stage had a much greater GA content than the MS1 and MS2 stages, suggesting that gibberellin may play a more major role in the late stage of male cone growth development ( Figure 4 ). Numerous investigations have demonstrated that BR is connected to flower growth development (Domagalska et al., 2007). For instance, Arabidopsis flowers later when the BR concentration is decreased (Shahnejat-Bushehri et al., 2016). The BR content was also significantly different (P<0.05) during the growth development of P. koraiensis’ female cones, and the accumulation was higher during the FS2 and FS3 phases, suggesting that it may played a key role in the later growth.

Based on morphological structure traits and physiological changes, the flowering process of plants is split into different stages. Currently, flowering determination, flower evocation and floral organ development are the three primary steps of a plant’s flowering process. According to earlier research, all flowering pathways are grouped together to form a regulatory network that interacts with one another to carry out each pathway’s specific regulatory function. The CO, FLC, LFY, SOC1, FY and FT genes are the key nodes of each regulatory convergence, among which the LFY, SOC1, FY and FT genes integrate the flowering pathway together (Komeda, 2004; Chen et al., 2022). The vernalization pathway has a significant impact on the timing of flowering in A. thaliana, and the genes AGAMOUS LIKE19 (AGL19), FLOWERING LOCUS T (FT), and FLC are key players in this system (Suter et al., 2014). While FCA and FY work together to control FLC RNA processing, FLC is a MADS-box transcription factor that controls the activity of genes necessary for meristem development from vegetative development to floral development (Marquardt et al., 2006; Rataj and Simpson, 2014). In conifers, the function of FT gene is still controversial (Klintenas et al., 2012). Studies have showed that FT plays a key role in the formation and growth of Norway spruce bud set, but is not involved in the induction of Pinus halepensis cone flowering induction, but is related to the vegetative bud dormancy (Gyllenstrand et al., 2007; Reisfeld et al., 2022). In this study, transcriptome sequencing of male and female cones produced 71097 and 31195 DEGs, respectively, that were enriched in different cone flowering process pathways. In the protein coexpression network, we discovered FT (Cluster-3272.97594, Cluster-6422.74394) in the male and female cones. More evidence that these genes may have distinct regulatory effects on the early growth of male and female cones came from the discovery that these genes were significantly strongly expressed in the MS1 stage. One of the most important variables in regulating flower growth development is phytohormone signaling, which controls a variety of morphogenesis processes in plants (Davis, 2009). In higher plants, the biosynthesis of GA is mainly divided into three stages, and the decibel is catalyzed in the plastid, endoplasmic reticulum and cytoplasm (Eshed and Lippman, 2019; Wu et al., 2020). In this work, 25 DEGs were found to be active in GA biosynthesis during the growth of male cones, with high levels in the MS1 and MS2 phases that encouraged the buildup of gibberellin ( Figure 8A ). The GA signaling pathway’s negative regulator is the DELLA protein. The expression of associated genes that are either repressed or boosted by DELLA varies as the concentration rises, causing the protein to be destroyed through the ubiquitination pathway and facilitating plant flowering (Bao et al., 2020). In this study, 11 DELLA genes were discovered in male cones, which may promote the ubiquitination of the DELLA protein. The megaspore mother cell (MMC) is the first female germ cell differentiated from flowering plants. After developing into female gametophytes, it provides fertilization sites for plants. Studies have shown that BR biosynthesis and signal transduction genes accumulate in the sporophytes of ovule primordia. By activating WRKY23 transcription factors, BR signaling influences the growth of megaspore mother cells. The BRI1 receptor and BZR1 transcription factor family primarily control this activation (Cai et al., 2022). This study revealed the expression of six BZR1 genes in the female cone BR biosynthesis pathway, which may be crucial to the growth development of female cones.

This study lays a strong foundation for understanding the regulatory mechanism controlling the growth development of P. koraiensis cones, even though more research is still required to fully understand the process of flower bud transformation and sex differentiation prior to the development of P. koraiensis cones.

In conclusion, after long-term phenological observation, we initially recorded the main developmental processes of male and female cones of P. koraiensis, and performed hormone content determination and transcriptome analysis on three crucial developmental stages. The results of hormone content determination showed that GA and BR were mainly accumulated in the late stage of development, indicating that they may be the key hormones to promote the development and maturation of male and female cones. Then, a total of 71,097 and 31,195 DEGs were identified in this research, which provided available genetic information for the study of the molecular breeding of P. koraiensis. In addition, this study focused on the hormone signal transduction pathway and biosynthesis pathway during the development of male and female cones, and evaluated and discussed them. Combined with transcriptome data, all transcription factors were screened out, and the expression levels of important genes MADS-box related to flowering were analyzed. Finally, the coexpression network was used to further predict that Cluster-3272.121832 and Cluster-6422.77906 may be the crucial regulatory genes for the development of male and female cones of P. koraiensis.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

YL: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. MZ: Methodology, Writing – review & editing. KC: Methodology, Writing – review & editing. LL: Software, Writing – review & editing. RH: Software, Writing – review & editing. XP: Data curation, Writing – review & editing. LZ: Data curation, Writing – review & editing. XZ: Data curation, Funding acquisition, Resources, Validation, Writing – review & editing.