Logical Operator Operator Search Text Search Type _add_circle_outline_ _remove_circle_outline_ [. Floral initiation, which precedes flower bud differentiation, represents a critical developmental stage affecting the flowering outcomes. However, the molecular mechanisms underlying floral initiation in C. oleifera remain poorly understood. In this study, buds from five key developmental stages of a 12-year-old C. oleifera cultivar ‘changlin53’ were collected as experimental samples. Scanning electron microscopy was employed to identify the stage of floral initiation. UPLC-MS/MS was used to analyze endogenous gibberellin (GA) concentrations, while transcriptomic analysis was performed to reveal the underlying transcriptional regulatory network. Six GA types were detected during floral initiation and petal development. GA 4 was exclusively detected at the sprouting stage (BII), while GA 3 was present in all samples but was significantly lower in BII and the flower bud primordium formation stage (BIII) than in the other samples. A total of 64 differentially expressed genes were concurrently enriched in flower development, reproductive shoot system development, and shoot system development. Weighted gene co-expression network analysis (WGCNA) identified eight specific modules significantly associated with different developmental stages. The magenta module, containing Unigene0084708 (CoFT) and Unigene0037067 (CoLEAFY), emerged as a key regulatory module driving floral initiation. Additionally, GA20OX1 and GA2OX8 were identified as candidate genes involved in GA-mediated regulation of floral initiation. Based on morphological and transcriptomic analyses, we conclude that floral initiation of C. oleifera is a continuous regulatory process governed by multiple genes, with the FT-LFY module playing a central role in the transition from apical meristem to floral meristem.
1. Introduction
Camellia oleifera Abel. (C. oleifera) is an evergreen tree species cultivated in subtropical regions and is one among the four major woody oil plants globally, along with oil palm, olive, and coconut. Because of geographical and climatic constraints, C. oleifera is widely distributed in the subtropical mountainous regions of the Yangtze River Basin. Its seeds yield tea oil rich in oleic acid and natural antioxidants, with notable nutritional and health benefits. Flowering is a critical agronomic trait affecting C. oleifera yield, and floral initiation plays a pivotal role in determining flowering time.
Floral initiation is influenced by both external and internal signals. Environmental factors, such as photoperiod, temperature, nutrient status, drought, salinity, exogenous hormones and chemicals, and microbial pathogens, as well as endogenous signals, such as plant age, hormone levels, and carbohydrate status, all contribute to its regulation. These cues are perceived primarily in the leaves and shoot apical meristem. In 1-year-old herbaceous plants, floral initiation marks the transition from vegetative to reproductive growth. Molecular studies on this process have largely focused on model herbs such as Arabidopsis thaliana and rice. In A. thaliana, flowering is regulated by a complex network involving more than 100 genes across multiple pathways: photoperiod, ambient temperature, autonomous, integrator, gibberellins (GAs), and vernalization. By contrast, floral initiation in woody perennials occurs in two distinct phases: the initial transition from vegetative to reproductive growth in the life cycle (marking maturity and the capacity to flower) and the annual initiation of floral structures post-maturity. Unlike annuals—where flowering is terminal and leads to senescence—perennials undergo repeated cycles of vegetative and reproductive growth. Hence, annuals and perennials have different flowering traits.
In Arabidopsis, the FT gene promotes flowering and is upregulated by CONSTANS (CO), which is sensitive to photoperiods. FT is repressed by FLOWERING LOCUS C (FLC), which delays flowering by blocking gene transcription in photoperiodic flowering. The attenuation of FLC, a key floral repressor, distinguishes summer- and winter-annual phenotypes. In perennial poplar, functionally differentiated FT1 and FT2 separate the timing of reproductive initiation and vegetative growth to different seasons. FT1 rises in response to low temperatures of winter and promotes reproduction, and FT2 is induced by exposure to long days and warm temperatures of spring and early summer and promotes vegetative growth. LEAFY (LFY), a primary activator of floral meristem development, plays a crucial role in determining floral transition timing. Its expression is promoted by SQUAMOSA PROMOTER BINDING-LIKE (SPL) transcription factors (e.g., SPL9) and GA-sensitive DELLA proteins. Interestingly, LFY expression is high in alternate-bearing mango (Mangifera indica L.) cultivars during flowering but inhibited in ever-flowering longan cultivar ‘Sijimi’. These examples underscore the mechanistic differences in floral initiation between woody and herbaceous plants. Due to long generation times and complex genetic backgrounds, the molecular mechanisms of flowering in woody perennials remain less explored than in model herbs.
In C. oleifera, flowering is essential for yield, yet its regulation is complex. Flower buds typically form at the base of vegetative buds located at the terminals of spring shoots or in the axils of new leaves. These spring shoots originate from vegetative buds formed in the previous year, which themselves are located at the terminals or leaf axils of older shoots. Flower bud differentiation in C. oleifera has been reported to occur between early April and late July, with flowering taking place from early October to the end of December. The timing of bud differentiation and flowering varies among cultivars. Floral initiation precedes flower bud differentiation and is a critical developmental stage influencing C. oleifera flowering. We revealed that old leaves in C. oleifera serve as key photoperiodically sensitive organs, promoting FT expression through light-responsive mechanisms to initiate floral bud formation. The transduction of endogenous signaling molecules from old leaves to axillary buds is required to initiate floral transition through induced transcriptional activation of floral meristem-specific genes. However, the specific genetic determinants mediating this developmental shift remain to be systematically characterized through comprehensive transcriptomic analysis, and the following question remains: which genes, LFY, APETALA1 (AP1) or any other genes, in the buds were activated?
2. Results
2.1. Morphological Characteristics of Floral Initiation in C. oleifera at Different Developmental Stages
The formation of flower bud primordia indicates that the floral meristem has been activated, and floral initiation has begun. Buds are key organs during floral initiation; therefore, buds at five key developmental stages were selected for transcriptome analysis: dormancy stage (stage I), sprouting stage (stage II), flower bud primordium formation stage (stage III), petal differentiation stage (stage IV), and completion of petal differentiation stage (stage V).
Figure 1. Developmental stages of buds in C. oleifera. Buds (a,b) are referred to the vegetative bud formed in the last year. Buds in stages I are distinguished as follows: (a) developed into sprouted buds (b); only vegetative buds grown (f,g); no flower buds occurred in stages I and II. Buds (c–e) in stages III, IV, and V were the apical buds of spring shoots (panel c–e), which were developed from a vegetative bud (b). Flower buds (h–j) in III, IV, and V were located at the base of apical buds (c–e). lp: Leaf primordium; fp: floral primordium; l: leaf, s: sepal; p: petal. The scale bars in a to b, in c to e, and in f to j indicate 1.5 cm, 1000 μm, and 50 μm, respectively.
2.2. Quantitative Analysis of Endogenous Gibberellins in Buds at Different Developmental Stages
To investigate the involvement of GAs in floral initiation, we quantified endogenous GA levels in C. oleifera buds across five developmental stages. Of the 10 GA types assessed (GA 1, GA 3, GA 4, GA 7, GA 9, GA 15, GA 19, GA 20, GA 24, and GA 53), only 6 GAs—GA 3, GA 4, GA 9, GA 15, GA 19, and GA 20—were detected (Figure 2a). The total GA content was significantly higher in stages BII, BIII, and BV than in stages BI and BIV (Figure 2a). The dominant GA types in each stage were as follows: GA 3 in BI and BV, GA 19 in BIV, and GA 20 in BII and BIII (Figure 2b). GA 3 and GA 4 are the biologically active forms of GA gibberellin. Notably, bioactive GA (GA 3 + GA 4) levels in BI, BII, BIII, and BIV were significantly lower than those in BV. GA 4 was exclusively detected in BII, while GA 3 was present in all stages, albeit at significantly lower levels in BII and BIII. These findings suggest that floral initiation in C. oleifera requires only a relatively low concentration of active GAs, whereas a higher concentration is essential for petal differentiation.
Figure 2. Concentrations of endogenous gibberellin types (a) and their relative proportions (b) at different developmental stages. The letters on the top of column indicate the multiple comparison results of total GAs concentration at 0.05 level.
2.3. Transcriptome Assembly and Annotation
Transcriptomic analysis using RNA sequencing (RNA-seq) was performed to investigate the gene regulatory networks underlying floral initiation across the five developmental stages of C. oleifera buds. An overview of the RNA-seq samples and clean read counts is shown in Table 1. The proportion of mapped reads per library ranged from 79.05% to 83.41%. Sequencing quality metrics were high, with Q30 values (sequences with a sequencing error rate of <0.1%) of >91% and Q20 values (sequ
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