1. Introduction
Camellia oleifera Abel. (
C. oleifera), a unique woody oil tree species endemic to China, is primarily found in the Yangtze River basin and the hilly regions south of China [
1,
2]. It is a late self-incompatibility plant. The flowering periods of different varieties are different, and pollination is difficult to complete. Moreover, its flowering period coincides with autumn and winter, a time when low temperatures and unpredictable weather conditions hinder flowering and pollination. Consequently, in practical production, there are more flowers and less fruit, a low fruit setting rate, and a low yield phenomenon [
3,
4]. Hence, investigating the flowering mechanism of
C. oleifera holds immense significance for enhancing its yield.
Photoperiod, the phenomenon that plants regulate their flowering time by sensing the duration of daylight and darkness, is a crucial factor influencing plants flowering and fruiting [
5]. The circadian clock, which functions as an endogenous timer that detects changes in day length during the photoperiodic response, is a primary pathway for photoperiodic flowering induction [
6,
7,
8,
9]. The
GI gene, situated between the circadian clock and flowering-regulating genes, serves as a circadian clock output gene [
9,
10]. In
Arabidopsis thaliana (
Arabidopsis), the
CONSTANS (
CO) gene plays an essential role in regulating flowering. It induces flowering in plants by promoting the expression of
FLOWERING LOCUS T (
FT), thereby [
11]. As one of the flowering regulatory factors,
GI regulates plants’ flowering by modulating the expression of the
CO and
FT genes under long-day conditions.
GI stands as an upstream gene of
CO and
FT, making it significantly important in regulating plant physiological rhythms and the flowering process [
12,
13,
14,
15].
In
Arabidopsis, the ZTL protein regulates the distribution and expression peak of the GI protein between the nucleus and cytoplasm through its N-terminal LOV domain, thus affecting the regulation of the biological rhythm and flowering time of
Arabidopsis [
16]. Through an analysis of the structure and function of
CmGI in chrysanthemums, it was found that
CmGI could be regulated in response to biological clock signals in the photoperiodic flowering pathway of chrysanthemums [
17]. In rice,
OsGI regulates the expression of three-quarters of genes associated with the circadian rhythm [
18]. Related studies on the circadian rhythm of Norwegian spruce showed that the functions of PaCCA1, PaGI, and PaZTL were similar to those of
Arabidopsis homologous isomerins [
19]. These results indicate that the function of
GI in regulating plant circadian rhythm may be highly conserved in different types of plants. Therefore, we speculate that the
CoGI gene is the key gene regulating the flowering time in
C. oleifera in response to the circadian rhythm. In this study, the CDS sequence of the
CoGI gene of
C. oleifera was obtained by screening the transcriptome sequencing results. After obtaining the full-length sequence, its function and potential interacting proteins were studied to verify its function in
C. oleifera and speculate the possible biosynthesis pathway.
4. Discussion
The GI protein has no known functional domain, and it is a higher plant-specific nuclear protein [
31]. In this study, we cloned the CDS sequence of
CoGI and conducted a translation as well as a bioinformatics analysis of its encoded protein. We found that the CoGI protein sequence was more than 80% similar to other species, indicating that GI was highly conserved among different species, which was consistent with previous studies.
It was found that
GI genes have tissue and organ specificity in different species. In
Arabidopsis,
AtGI exhibits elevated expression levels in leaves, flowers, and stems, with higher expression observed in stems compared to roots [
32]. In
Glycine max, the expression of
GmGI was the highest in leaves and flower buds under a long-day condition. However, under short-day conditions,
GmGI expression peaked in roots and leaves [
33].
BnGI was expressed in all tissues of
Brassica napus, with significantly higher levels in rapeseed compared to other tissues [
34]. In
Dioscorea esculenta,
DeGI expression was higher in leaves and roots, but lower in stems [
35]. In
Solanum tuberosum,
StGI was highly expressed in roots, stolons, and sepals [
36]. In
Citrus reticulata Blanco,
CrGI expression peaked in leaves, followed by stems [
37]. Similarly, in
Juglans regia,
JrGI expression was significantly higher in leaves compared to leaf buds and flower buds [
38]. In this study,
CoGI expression in different tissues of
C. oleifera was analyzed and
CoGI was found to be highest in leaves. Previous studies have shown that the circadian clock components control leaf senescence in
Arabidopsis, and GI positively regulates leaf senescence [
14,
39,
40,
41]. In addition, the highest expression of
GI in plant leaves may also be because leaves are the most sensitive organs for sensing photoperiods and responding to biorhythmic clock signals.
GI has many functions and effects in regulating flowering time. In the study of
Arabidopsis, it was discovered that AtGI can interact with FLAVIN-BINDING, KELCH REPEAT, and F-BOX 1 (FKF1) proteins to form the ATGI-FKF1 complex under long-day conditions, degrade CDF1 protein on
CO promoter, and release the inhibition of
CO. Thus, it can promote the expression of its target gene
FT and promote the flowering of
Arabidopsis [
42,
43,
44,
45,
46]. However, under short-day conditions,
GI can enhance the expression of
FT without up-regulating
CO expression, thus promoting the flowering of
Arabidopsis [
47]. Moreover, in the relevant studies on the long-day monocotyledonous plant
Brachypodiam distachyon, it was found that the function of
BdGI to induce flowering was the same as that in
Arabidopsis [
48]. In this study, we transferred the
CoGI into Col-0
Arabidopsis plants to obtain transgenic
CoGI-overexpressing
Arabidopsis plants. It was found that transgenic plants exhibited significantly earlier flowering time compared to Col-0
Arabidopsis plants, and the number of rosette leaves was significantly reduced during bolting.
Although many proteins interacting with
GI genes have been reported in
Arabidopsis, it is not clear which proteins CoGI binds to and influences the expression in
C. oleifera. Therefore, we conducted yeast two-hybrid experiments to find the proteins binding to CoGI and explore the response mechanism of CoGI in the
C. oleifera flower formation from the perspective of genetic regulation. One of them, the
ACR9 gene encodes a protein containing ACT domain repeats. In rice, the distribution and accumulation of the OsACR9 protein show dynamic changes in different tissues and developmental stages, especially in the parts where nitrogen N-metabolism is active. For example, the accumulation of OsACR9 protein in rice root cells increases significantly after the application of ammonium salt [
49]. These results indicate that it is involved in the regulation of N-metabolism. In
Arabidopsis,
acr9 mutants are highly sensitive to glucose, especially in early seedling development, root growth, anthocyanin accumulation, and significantly increased expression of sugar response genes, suggesting that
ACR9 is a negative regulatory component in glucose signaling pathways. At the same time,
acr9 mutants are also hypersensitive to Abscisic Acid (ABA), suggesting that
ACR9 may also play an inhibitory role in the ABA signaling pathway and may mediate the interaction between C/N signals [
50]. At4g27190 is a protein with potential disease resistance in plants. In a study of the durian plant, a large number of resistance gene analogs (
RGAs) were identified through the mining and analysis of the reference genome of the durian “Musang King”, among which 135 copies of the disease resistance protein encoded by
At4g27190 were found in the durian genome. Suggesting that it may play an important role in durian defense responses to a variety of pathogens [
51]. In comparative proteomic studies during lychee flowering, homologous proteins encoded by
At4g27190 were specifically expressed in the phloem exudate at the start-up stage of flower buds, and were associated with signal transduction, hormone-mediated signaling pathways, plant response to ABA and other biological processes [
52].
GI is not only related to glucose metabolism in plants, but also plays a key role in the photoperiodic signal response to drought stress.
GI can also activate
FT/
TSF and
SOC1 genes in response to ABA, resulting in early flowering.
The UPF0481 protein has been shown to have many possible functions in plants, including stress response, tissue culture, and regeneration processes. In the study of
Elsholshia haichuensis, the UPF0481 protein was differentially expressed between copper-resistant and non-copper-resistant populations, indicating that this protein may play a role in plant response to heavy metals (such as copper) stress [
53]. In the study of eucalyptus, the homologous gene of this protein showed dynamic expression changes during callus development. This suggests that it also plays a role in dedifferentiation, redifferentiation and developmental regulation of plant cells [
54]. IDM3 protein plays multiple roles in plant DNA methylation regulation. It is not only a core component of the
ROS1-mediated DNA demethylation pathway, but also a key factor involved in histone acetylation regulation. In
Arabidopsis,
IDM3 interacts with proteins such as
IDM1,
IDM2, and
MBD7, and this complex can trigger histone acetylation modification targeting specific methylation regions, thus initiating the recruitment of demethylases such as
ROS1 and achieving DNA demethylation [
55,
56]. In
Camellia sinensis,
CsIDM3 also interacts with MBD family members
CsMBD5 and
CsMBD16, suggesting that IDM family members may regulate the epigenetic silencing of specific methylation regions by interacting with MBD proteins [
57].
LAO is a copper-containing oxidase in plant cells, mainly located in the cytoplasm or near the cell wall, which participates in terminal oxidation reactions, and can catalyze the oxidation of ascorbic acid (vitamin C) to dehydroascorbic acid, a process that is crucial for material metabolism in plants, especially playing a central role in regulating the REDOX state outside the plastid [
58]. As an antioxidant and signaling molecule, ascorbic acid concentration fluctuates under strong light environment, which may be related to plant adaptation to light intensity change, regulation of the photoprotection mechanism and coping with oxidative stress. Studies have shown that under intense light conditions, ascorbic acid concentration in plant leaves changes regularly during the day, and is closely related to the establishment of flowering time, defense response to stress, and expression regulation of genes related to plant hormone signaling [
59,
60,
61]. The above three proteins are involved in plant stress response, DNA methylation, and antioxidant synthesis, etc. Although no studies have proven that
CoGI has a direct relationship with these three enzymes,
GI genes play an important role in plant stress response. In
Arabidopsis, the activity of
DNA MENTHYLTRAN-SFERASE 1 (
MET1) is coordinated with the oscillation of the biological clock, which affects the methylation state of genes related to circadian rhythms, and thus regulates the expression of these genes [
62]. Moreover, the daily regular variation of ascorbic acid concentration in plant leaves indicates that it may be influenced by circadian rhythms.
At present, there are few reports about DExH12-like and IT1K-like in plants, and the functions of these two plant proteins and the biological processes involved in them are limited. Therefore, their mechanisms of action and regulatory pathways were not discussed in depth in this study.