Photoperiod-entrained circadian regulation of downstream transporter networks mediates nyctinastic leaf movement in peanut

Abstract

The nightly closure or downward folding of leaves, a process known as sleep movement or nyctinasty, is a widespread phenomenon in legumes that helps optimize photosynthesis, conserve water, and reduce pathogen susceptibility. Although the physical process has been well characterized, the molecular regulation of nyctinasty remains poorly understood. Here, we combined temporal transcriptome profiling, photoperiod manipulation, and tissue-specific gene expression analysis to dissect the regulatory network in peanut (Arachis hypogaea) during nyctinastic leaf movement. Under normal light/dark cycles, leaves displayed distinct nyctinastic behavior, which was suppressed under continuous light, underscoring the importance of photoperiod for this process. Transcriptome analysis identified conserved light- and circadian-related genes exhibiting rhythmic expression patterns. Key candidates included light-responsive SWEET and aquaporin genes with distinct tissue-specific expression patterns, along with a pulvinus-enriched MYB transcription factor gene. Promoter-binding and reporter assays supported transcriptional links between circadian/light-signaling components and these candidate downstream genes. Together, these findings support a working model in which photoreceptors and the circadian clock coordinate the expression of transporter and regulatory genes, which may contribute to nyctinastic leaf movement in peanut. These findings provide a molecular framework for future functional investigations of nyctinasty in legumes.

1. Introduction

Circadian regulation in plants is an endogenous rhythmic phenomenon driven by the circadian clock, which is primarily composed of transcription–translation feedback loops. The core oscillator includes genes such as Late Elongated Hypocotyl/Circadian clock associated 1 (LHY/CCA1), members of the Pseudo-response regulator (PRR) family, Timing of CAB expression 1 (TOC1), and the Lux arrhythmo (LUX)–Early flowering 3 (ELF3)–ELF4 complex. Light signals are integrated into the circadian network through photoreceptors such as phytochromes and cryptochromes. This regulatory mechanism coordinates outputs such as photosynthesis, stomatal movement, and stress-responsive gene expression, playing significant roles in plant adaptation to photoperiod and crop improvement [1].

Leaf movements in leguminous plants, including peanut (Arachis hypogaea), represent a striking example of circadian-regulated physiology [2]. Like many other legumes, peanut exhibits the nightly closure or downward folding of leaves, a process known as nyctinasty or “sleep movement”. The opening of peanut leaflets during the day and their closure at night follow a clear diurnal rhythm, with leaflets opening to enhance photosynthetic efficiency in the daylight and closing at night to conserve water and reduce the risk of herbivory [3]. The leaf movement is achieved primarily by changes in turgor pressure within specialized cells called pulvini located at the base of the leaflet. These cells undergo changes in volume due to changes in ion fluxes and water content, causing the leaflets to move [4].

Nyctinasty, the nightly closure or downward folding of leaves, is a widespread, convergently evolved behavior in plants. This trait has been documented across diverse lineages, including numerous legumes [3]. Multiple hypotheses have been proposed to explain why nyctinasty evolved, which are not mutually exclusive. One key advantage is improved water balance and temperature regulation. By folding leaves vertically at night, plants can reduce radiative heat loss and prevent excessive cooling or frost damage to the leaf surface [5,6]. Nocturnal closure also reduces the exposure of the stomata-rich leaf surface, thereby limiting nighttime transpiration and conserving water, a benefit particularly important for plants in arid or xeric environments [3]. Another benefit of leaf closure is the shedding of water: folded leaves tend to channel dew or rain off the leaf surface, keeping the leaflet drier and making it less hospitable to fungal pathogens. Indeed, peanut leaves with greater nocturnal leaf angles (less horizontal surface) had lower wettability and were less susceptible to fungal rust infection than those with smaller nocturnal leaf angles [7]. Thus, nyctinasty in peanut and other plants likely mitigates moisture-related stresses, balancing hydration and pathogen defense.

The rhythmic opening and closing of leaves is driven by the circadian clock and environmental cues such as light and temperature [8,9]. The molecular underpinnings include complex interplay between circadian rhythms, environmental signals, and phytohormones [10,11]. Key components include circadian clock genes [12], which help synchronize leaf movements with the day–night cycle, and signal transduction pathways involving multiple ions and reactive oxygen species that mediate changes in turgor of the pulvini [4,13]. Photoreceptors such as phytochromes and cryptochromes perceive light signals and initiate a cascade of molecular events that influence the circadian clock and downstream effectors [14]. Circadian clock genes interact with environmental signals to fine-tune leaf movements. For example, phytochromes and cryptochromes relay light information to the circadian clock, ensuring that leaf movements are synchronized with the external light–dark cycle. This coordination is crucial for optimizing photosynthetic efficiency and water use during the day and night [15]. The morning-expressed genes LHY and CCA1 initiate the daytime phase of the circadian cycle. These genes encode transcription factors that repress the expression of evening-phased genes, including TOC1. In turn, TOC1 accumulates and inhibits the expression of LHY and CCA1, thereby maintaining the oscillatory nature of the circadian clock [16].

In addition to photoreceptors and circadian clock genes, other key genes involved in this process include those encoding ion transporters, aquaporins, and components of the signaling pathways that modulate changes in turgor pressure [17]. Three key ion channels have been identified in the mimosoid tree Samanea saman: the slow-type anion channels SLOW ANION CHANNEL-ASSOCIATED 1 HOMOLOG 1 (SsSLAH1) and SsSLAH3 and the potassium channel SHAKER-TYPE POTASSIUM CHANNEL 2 (SPORK2). The cell-specific circadian expression of SsSLAH1 plays a crucial role in nyctinastic leaf opening [18]. Moreover, in tobacco (Nicotiana tabacum) plants, aquaporins exhibit diurnal oscillations in expression within petioles, aligning with the leaf expansion patterns. Protoplasts isolated from tobacco petioles showed higher cell water permeability in the morning (during the leaf unfolding phase) and lower permeability in the evening. Additionally, transgenic tobacco lines with impaired Aquaporin 1 (NtAQP1) expression demonstrated reduced diurnal leaf movements. Therefore, the diurnal oscillatory expression of aquaporins is a crucial component of the mechanism driving leaf movements [19].

Although the mechanisms controlling leaf movements in legumes have been extensively studied, the key genes and the complete mechanisms remain unclear. Specifically, in peanut, an important legume species, the genetic and molecular basis of these leaf movements is not well understood. Such information could increase our understanding of how plants adapt to their environment and optimize their physiological processes. In this study, we explored the gene regulatory landscape of peanut leaf movements in response to photoperiod. By analyzing transcriptome data from compound leaves at different times of the day and night, we identified key genes and regulatory networks that might be involved in this photoperiodic response. Our findings enhance our understanding of the molecular mechanisms governing leaf movements, with broader implications for plant adaptation and productivity.

2. Results

2.1. Peanut leaf movements exhibit distinct responses to different light conditions

Under an 8 h dark/16 h light cycle, leaves were fully open at 21:00 before lights off, partially closed by 22:00, and completely closed by 23:00. Upon light exposure at 5:30, the leaves were half open by 6:00 and fully open by 7:00 (Fig. 1A and Video 1). This rhythmic movement points to the strong photoperiodic regulation of leaf nyctinasty. By contrast, plants under continuous light maintained a fully open leaf position throughout the 24-h period, exhibiting minor up-and-down movements but without the characteristic opening and closure observed under normal light/dark cycles (Video 2). This finding indicates that light is a dominant factor regulating peanut leaf movements, with the absence of darkness preventing leaf closure.

Fig. 1.

Changes in diurnal leaf movement and photosynthesis in peanut leaves under different light conditions. A Peanut plants were subjected to either a normal photoperiod of 16 h light/8 h dark (top panel) or continuous light for 24 h per day (bottom panel) for seven days. Under a normal photoperiod, the lights were switched on at 05:30 and switched off at 21:30. The images illustrate the dynamic changes in leaf position over time. B Effects of light conditions on photosynthetic and gas exchange parameters in peanut. Data are presented as the mean ± standard error of six biological replicates.

To investigate the effects of photoperiod on photosynthetic performance, we analyzed gas exchange parameters under normal (16 h light/8 h dark) and continuous light (24 h light) conditions (Fig. 1B). The net photosynthetic rate exhibited a distinct diurnal rhythm under a normal photoperiod, peaking around 12:00 p.m., followed by a decline. However, under continuous light, the peak was reduced, suggesting that the rhythmic regulation of photosynthesis was disrupted. Stomatal conductance and the transpiration rate showed similar temporal patterns, with pronounced peaks under normal light/dark conditions but lower peaks under continuous light. By contrast, intercellular CO₂ concentration showed an inverse trend, decreasing during peak photosynthesis under normal conditions but remaining relatively stable under continuous-light conditions. These findings indicate that prolonged exposure to light alters stomatal behavior and photosynthetic efficiency in peanut, likely due to a disruption of the circadian rhythm and impaired stomatal regulation.

2.2. Changes in light signaling–related gene expression in peanut

Light serves as a primary external cue modulating diurnal leaf movement, suggesting that photoreceptors and the circadian clock play crucial roles in regulating leaf positioning in response to environmental light cycles. We therefore examined the changes in expression of light signaling–related genes and circadian clock-associated genes under different conditions.

Phylogenetic analysis identified key light-responsive genes, including Phytochrome (PHY), Cryptochrome (CRY), Phototropin 1 (PHOT1), Constitutively photomorphogenic 1 (COP1), Suppressor of phyA-105 (SPA), UV resistance locus 8 (UVR8), Elongated hypocotyl 5 (HY5), and Phytochrome-interacting factors (PIFs), along with their conserved domain structures (Fig. S1A). We analyzed the expression profiles of these genes under normal (8 h dark/16 h light) and continuous light (24 h light) conditions across multiple time points (Fig. S1B). Highly expressed genes exhibited elevated expression levels, suggesting they might function as key regulators of light signaling in peanut. The fluctuations in gene expression detected in this analysis indicate that many of these genes exhibit light-responsive rhythmic patterns, with some reaching peak expression during the light phase and becoming downregulated in the dark (Fig. S1B).

2.3. Changes in circadian clock-associated gene expression in peanut

Similarly, we examined the expression patterns of circadian clock-associated genes in peanut. Phylogenetic analysis identified key circadian regulators along with their conserved domain structures (Fig. S2A). We examined the expression profiles of these genes under normal (8 h dark/16 h light) and continuous light (24 h light) conditions across multiple time points (Fig. S2B).

We further analyzed the dynamic expression trends of representative, highly expressed genes from each gene family (Fig. 2A). Considering the similar expression profiles between the A and B subgenomes of the allotetraploid plant peanut, we analyzed the expression dynamics of key genes from each family using subgenome A as a representative (Fig. 2A); the parallel expression patterns for subgenome B are shown in Fig. S2C. Unlike the expression patterns observed for light signaling–related genes, most circadian clock-related genes showed peak expression around light–dark transitions. Exceptions included chr06.3134_ELF3 and chr01.3196_TOC1, whose expression peaked at night, while chr02.374_LHY/CCA1 and chr02.1648_RVE8 expression peaked during the day (Fig. 2A). Although continuous light markedly reduced the oscillation amplitude of expression of several clock-associated genes, in general, their relative temporal ordering remained unchanged. Morning-phased genes, such as RVE8 and LHY/CCA1, and evening- or night-phased genes, such as TOC1 and ELF3, retained generally distinct expression phases rather than becoming completely disorganized. Thus, continuous light appeared to attenuate, rather than entirely abolish, the temporal organization of clock-associated gene expression. Notably, the pronounced opening–closing movement of peanut leaves disappeared under continuous light, suggesting that molecular clock-associated transcription and leaf movement may respond differently or become partially uncoupled under this condition. However, because we only examined one subjective cycle after seven days of continuous-light treatment, these data do not confirm the presence of sustained free-running circadian rhythmicity, a concept that will require multi-day temporal analysis. The strong consistency between spreading-type and erect-type cultivars further validates the results of transcriptomic analysis, reflecting a high degree of conservation in light-signaling pathways across different peanut germplasms.

Fig. 2.

Diurnal expression patterns of circadian clock-associated genes in peanut under normal and continuous light conditions. A Diurnal expression patterns of representative circadian clock-associated genes. The data shown are from subgenome A, while the corresponding expression patterns for subgenome B are provided in Fig. S2C. Expression levels (FPKM values) of selected representative genes in peanut under normal conditions (N: 8 h dark/16 h light) and continuous light treatment (T: continuous light, 24 h light). The red lines represent peanuts (S), while the blue lines represent erect-type peanuts (E). Sampling times are indicated on the x-axis as time of day. Under a normal photoperiod, lights were switched on at 05:30 and switched off at 21:30, as indicated by the sun and moon symbols, respectively. The light and dark phases are represented by white and shaded gray backgrounds, respectively. Data are presented as the mean ± standard error of three biological replicates. ELF3: Early flowering 3; ELF4: Early flowering 4; GI: Gigantea; LHY/CCA1: Late elongated hypocotyl/Circadian clock associated 1; LUX: Lux arrhythmo; PRRs: Pseudo-response regulators; RVE8: Reveille 8; TOC1: Timing of CAB expression 1; ZTL: Zeitlupe. B Timing of the peak expression of circadian clock-associated genes in peanut over a 24-h cycle. The timeline at the bottom represents a full diurnal cycle, with the light phase (5:30–21:30) and dark phase (21:30–5:30) indicated by sun and moon symbols, respectively. C Yeast one-hybrid assay examining the regulatory interactions between key circadian clock genes in peanut. LHY/CCA1 (morning component, chr02.374), PRR (afternoon component, chr02.360, peaking at 14:30), and TOC1 (night component, chr01.3196, peaking around 21:30). The symbols “+” and “−” indicate the presence or absence of interactions, respectively. The AbA concentration used in each Y1H assay is shown below the corresponding panel.

Mapping the peak expression times along the daily cycle revealed a sequential pattern: Reveille 8 (RVE8) expression peaked 30 min to 1 h after lights on; LHY/CCA1 expression peaked around 2 h after lights on; the expression of three Pseudo-response regulator (PRR) genes peaked at 14:30; the expression of Zeitlupe (ZTL), one PRR, and Early flowering 4 (ELF4) peaked at 18:30; Gigantea (GI), TOC1, another PRR, and LUX expression peaked around 21:30 (lights off); and ELF3 expression peaked approximately 1 h after lights off (Fig. 2B). These expression patterns are largely consistent with previous findings in other species [14,20].

Given the well-established regulatory relationships among circadian clock components in other plant species, we investigated the potential interactions among LHY/CCA1 (morning component, chr02.374), PRR95 (afternoon component, chr02.360, peaking at 14:30), and TOC1 (night component, chr01.3196, peaking around 21:30) in peanut via yeast one-hybrid (Y1H) assays. LHY/CCA1 specifically bound to the PRR95 promoter, whereas TOC1 bound to the LHY/CCA1 promoter (Fig. 2C). Dual-luciferase assays demonstrated that CCA1 repressed PRR95 promoter activity and that TOC1 repressed CCA1 promoter activity (Fig. S2D). Together, these results support conserved negative regulatory relationships among core circadian clock components in peanut and suggest that CCA1, PRR95, and TOC1 participate in a coordinated temporal regulatory network.

2.4. Validation of RNA-Seq data using RT-qPCR

To validate the reliability of the transcriptome data, we selected a set of light- and circadian clock-related genes for reverse transcription-quantitative PCR (RT-qPCR): LHY (chr12.452), CRY (chr17.2570), SPA (chr18.1607), PIF (chr17.449), PRR9 (chr02.360 and chr12.437), TOC1 (chr11.2021), RVE8 (chr12.1306), ZTL (chr13.4202), ELF4 (chr17.1457), and GI (chr19.3083). The expression trends observed by RT-qPCR closely matched those from the RNA-Seq data across various time points and conditions, confirming the diurnal rhythmicity of these genes and supporting the accuracy of the transcriptome data as a reliable representation of gene expression dynamics in peanut leaves (Fig. S3).

2.5. Co-expression analysis links light and circadian regulators with transporter-associated pathways in peanut

Given the identification of numerous light- and circadian rhythm-related genes in peanut, we looked for genes with similar expression patterns to these key regulators, as such genes may share close regulatory relationships and coordinate with diurnal leaf movement. We grouped genes with FPKM values > 20 under normal conditions (8 h dark/16 h light) in both spreading-type and erect-type peanuts into clusters based on expression patterns. Each peanut type was grouped into eight distinct expression clusters (Fig. 3A).

Fig. 3.

Co-expression analysis of light- and circadian-related genes in peanut. Time points 1–14 correspond to the time period from 5:30 to 2:30 the next day, as shown in Fig. 1, Fig. 2. Sampling times are indicated on the x-axis as time of day. Under a normal photoperiod, lights were switched on at 05:30 and switched off at 21:30, as indicated by the sun and moon symbols, respectively. The light and dark phases are represented by white and shaded gray backgrounds, respectively. A Expression clustering of genes with FPKM > 20 in spreading-type (upper) and erect-type (lower) peanuts under normal conditions. Eight clusters were generated for each plant type, showing normalized expression profiles. B Heatmaps showing the expression patterns of light- and circadian-related genes across different time points under normal conditions in both types of peanut. Light- and circadian-related genes are highlighted according to their cluster assignments.

By examining the clusters containing light- and circadian-related genes, we observed strong consistency between the two types of peanut, despite slight differences in specific gene clustering (Fig. 3B). For example, in both spreading-type peanuts under normal light/dark conditions (SN) and erect-type peanuts under these conditions (EN), most light-related genes were grouped into cluster 1. Most circadian rhythm-related genes were grouped into cluster 2 for SN and cluster 8 for EN. TOC1 and ELF3 were grouped into cluster 7 in SN and cluster 5 in EN. Notably, some light- and rhythm-related genes were grouped in the same cluster. For instance, PHOT1 (chr01.746) clustered with two PRR genes in SN, and CRY (chr09.1815) consistently clustered with circadian-related genes (ZTL, GI, ELF4, and three PRR genes) in both SN and EN, suggesting a strong connection between CRY and circadian regulation. To further characterize these relationships, we analyzed the intersecting genes of the corresponding clusters between the two types of peanut: SN_Cluster1 vs. EN_Cluster1 (light-related gene cluster), SN_Cluster2 vs. EN_Cluster8 (circadian rhythm-related gene cluster), and SN_Cluster7 vs. EN_Cluster5 (TOC1 and ELF3 cluster).

We conducted gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of these overlapping gene sets (Fig. S4). Genes from the light-related clusters were significantly enriched in light response and circadian rhythm pathways based on both GO and KEGG analyses. The transporter pathway was also highly enriched (ranking third in KEGG analysis), suggesting a possible link between transport processes and photoperiodic regulation. Genes from the circadian-related clusters were predominantly enriched in RNA processing, translation, and ribosome-related pathways, indicating that both transcriptional and translational regulation are actively involved in circadian control. We also performed GO and KEGG enrichment analyses of overlapping genes from SN_Cluster7 and EN_Cluster5, which include key circadian genes such as TOC1 and ELF3. These genes were significantly enriched in protein metabolism, RNA processing, and vesicle-mediated transport pathways. These results suggest that transcriptional regulation and intracellular transport processes may be closely linked to circadian regulation in peanut (Fig. S4).

2.6. Aquaporin and SWEET genes exhibit diurnal expression rhythms

The significant enrichment of the transporter pathway among genes from the light-related clusters attracted our interest. We identified 103 genes in the transporter pathway (Table S1) and selected 37 genes with an FPKM value > 100 in at least one of the 56 samples across SN, ST (spreading-type continuous light treatment), EN, and ET (erect-type continuous light treatment) for further analysis. Notably, chr07.897, an aquaporin gene, exhibited particularly high expression, with a clear diurnal rhythm that peaked during the daytime (Fig. S5A).

Next, we focused on 32 transporter genes from the same set that were upregulated after lights on (defined as SN14/SN1 < 0.5 and EN14/EN1 < 0.5) and classified them into the following functional categories: vacuolar amino acid transporters, ammonium transporters, potassium channels, magnesium transporters, ascorbate ferrireductases, MATE efflux family proteins, auxin efflux carriers, aquaporins (MIP family), mitochondrial carriers, sugar transporters, NRT1/PTR family proteins, and sulfate transporters (Fig. S5B). These genes consistently showed higher expression during the daytime and lower expression at night. After continuous-light treatment, the rhythmic oscillation of these genes was weakened to varying degrees, with a noticeable reduction in rhythmicity among three sugar transporter genes and two sulfate transporter genes.

Aquaporins are transporters whose members regulate diurnal leaf movement by altering water flux in motor tissues [21]. Although direct evidence linking SWEET genes to leaf movement is limited, sugar signaling and distribution can affect turgor pressure and energy availability, which are crucial for pulvinar movement [22]. Based on these insights, we further analyzed the expression patterns of aquaporin and SWEET genes to explore how light regulates their activity.

We identified the aquaporin and SWEET gene families in peanut based on homology with the corresponding genes in Arabidopsis (Arabidopsis thaliana). We identified 84 aquaporin genes belonging to four subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic proteins (NLMs), and small basic intrinsic proteins (SIPs). Additionally, we identified 49 SWEET genes in peanut, which we classified based on their similarity to AtSWEET genes. These genes displayed conserved domains typical of the major intrinsic protein (MIP) and MtN3/saliva (MtN3_slv) superfamilies, respectively, suggesting that they might play conserved roles in membrane transport processes (Fig. S6).

To explore the involvement of aquaporin and SWEET genes in light-related regulatory networks, we analyzed the overlapping genes from the light-related clusters (SN_Cluster1 vs. EN_Cluster1), which contained 1706 shared genes (Fig. S4). Within this gene set, we identified several aquaporin genes (primarily from the PIP subfamily) and SWEET genes (Fig. 4A). Most of these genes exhibited clear diurnal rhythmic expression patterns, characterized by higher expression levels during the daytime and lower expression levels at night under normal conditions. In particular, four SWEET genes and six PIP genes (marked with orange stars in Fig. 4A) showed strong light-responsive expression patterns, identifying them as candidate transporter genes potentially associated with turgor- and metabolite-related processes during leaf movement. These expression patterns suggest that these genes respond to changes in photoperiod, warranting further functional investigation.

Fig. 4.

Expression patterns of SWEET and aquaporin genes from the light-related gene cluster in peanut. A Expression profiles of SWEET and aquaporin (PIP) genes from the overlapping genes of SN_Cluster1 and EN_Cluster1 under normal (N) and continuous light (T) conditions in spreading-type (S) and erect-type (E) peanuts. Orange stars indicate the four SWEET genes and six PIP genes that exhibited strong light-responsive rhythmic expression patterns that were selected for subsequent analyses. B Smoothed temporal expression trajectories of selected SWEET, aquaporin, and transcription factor genes from the light-related gene cluster in spreading-type (SN) and erect-type (EN) peanuts under normal light/dark conditions. The expression profiles across the 14 sampling time points were ordered according to the actual collection times, from 05:30 to 02:30 the next day, and the sequential positions were linearly rescaled from 0 to 10 for visualization. Thus, the x-axis represents normalized temporal progression through the sampling sequence rather than the actual time on the clock. The y-axis represents the expression level of each gene, and the color gradient indicates the normalized temporal position. Orange stars indicate genes selected to analyze tissue-specific expression and regulation.

In addition, we identified key transcription factor (TF) genes within the same light-related clusters, which may serve as critical regulators of leaf activity. Among these were well-known TF genes involved in the circadian clock and light signaling, including LHY/CCA1, PIF, MYB, ERF, and G2-like family members (Fig. 4B). Pseudotime trajectory analysis revealed coordinated dynamic expression patterns between these TFs and the SWEET and aquaporin genes across the light/dark cycle, pointing to a transcriptional association between these TFs and transporter genes. These findings identify a candidate regulatory network that may link light and circadian signaling with transporter gene expression during peanut leaf movement.

2.7. Candidate regulatory genes exhibit issue-specific expression and light-responsive promoter activities

To investigate the spatiotemporal expression dynamics of genes potentially involved in nyctinastic leaf movement, we conducted RT-qPCR of selected SWEET sugar transporter, aquaporin, and key transcription factor genes across root, stem, leaf, compound leaf pulvinus, and leaflet pulvinus tissues at three time points (10:00, 16:00, and 21:00) (Fig. 5A).

Fig. 5.

Tissue- and time-specific expression of key light-responsive genes in peanut. A Relative expression levels of genes encoding SWEET sugar transporters (chr08.2947, chr07.2532), aquaporins (chr03.178, chr08.1447), and light/circadian-related transcription factors (LHY/CCA1: chr02.374, chr06.148; MYB: chr15.1144, chr15.2534; PIF: chr07.456) across root, stem, leaf, compound leaf pulvinus, and leaflet pulvinus tissue collected at three time points: morning (10:00), afternoon (16:00), and evening (21:00). Data are presented as the mean ± standard error of three biological replicates. B Dual-luciferase reporter assays assessing the promoter activities of SWEET (ProSWEET_07.2532, ProSWEET_08.2947), PIP aquaporin (ProPIP_03.178, ProPIP_08.1447), CCA1 (ProCCA1_02.347, ProCCA1_06.148), MYB (ProMYB_05.1144, ProMYB_15.2534), and PIF (ProPIF_07.456) in Nicotiana benthamiana leaves under light/dark conditions.

Most genes exhibited diurnal rhythmicity consistent with our transcriptome data, characterized by peak expression in the morning, reduced expression in the afternoon, and the lowest expression levels at night. SWEET genes (chr08.2947, chr07.2532) and circadian/light-related transcription factor genes (LHY/CCA1: chr02.374, chr06.148; MYB: chr15.1144; PIF: chr07.456) were preferentially expressed in leaves. By contrast, aquaporin genes (chr03.178, chr08.1447) were predominantly expressed in compound leaf pulvini, suggesting their involvement in turgor-driven leaflet movement. Importantly, the MYB gene chr15.2534 was preferentially expressed in the pulvini of both compound leaves and leaflets, identifying it as a candidate pulvinus-enriched regulator that may coordinate light and circadian signals with the expression of movement-associated downstream genes (Fig. 5A).

To investigate the transcriptional regulation of SWEET, aquaporin, and TF genes in peanut, we performed dual-luciferase reporter assays in Nicotiana benthamiana leaves. We detected significant light-responsive activity for the promoters of SWEET (ProSWEET_07.2532, ProSWEET_08.2947), PIP aquaporin (ProPIP_03.178, ProPIP_08.1447), LHY/CCA1 (ProCCA1_02.347, ProCCA1_06.148), MYB (ProMYB_05.1144, ProMYB_15.2534), and PIF (ProPIF_07.456). Luciferase imaging revealed that these promoters showed higher activity in the illuminated half of the leaf compared to the shaded half, indicating that they are transcriptionally activated by light (Fig. 5B).

2.8. LHY/CCA1 governs the expression of key genes, including MYB transcription factor aquaporin, and SWEET transporter genes

To further clarify the transcriptional regulatory relationships between key transcription factors and SWEET and PIP genes, we examined the potential roles of circadian- and light-related TFs in regulating the expression of these genes using a dual-luciferase reporter assay (Fig. 6A). Two MYBs (MYB-15.2534 and MYB-08.1369) and PIF-07.456 significantly induced SWEET_07.2532 promoter activity; two MYBs (MYB-05.1144 and MYB-08.1369) significantly induced SWEET_08.2947 promoter activity; and CCA1 (CCA1-02.347) and three MYBs (MYB-05.1144, MYB-08.1369, and MYB-15.2534) significantly induced PIP_08.1447 promoter activity. These findings suggest that circadian clock- and light-responsive transcription factors modulate SWEET and aquaporin gene expression, potentially influencing turgor-driven leaf movement by modulating sugar and water transport.

Fig. 6.

Transcriptional regulation of SWEET and aquaporin genes by light-responsive transcription factors in peanut. A Transcriptional regulation of SWEET and aquaporin gene promoters by circadian- and light-responsive transcription factors, as determined using dual-luciferase reporter assays. Data are presented as the mean ± standard error of six biological replicates. Differences among treatments were evaluated using one-way ANOVA followed by the specified post hoc multiple-comparison test. Statistical comparisons were made between each transcription factor treatment and the corresponding empty-vector control. Double asterisks indicate significant differences at P ≤ 0.01. B Yeast one-hybrid (Y1H) assays validating the direct binding of transcription factors to the PIP_08.1447, SWEET_07.2532 and SWEET_08.2947 promoters. The symbols “+” and “−” indicate the presence or absence of interactions, respectively. The AbA concentration used in each Y1H assay is shown below the corresponding panel.

Y1H assays confirmed direct interactions between key transcription factors and the promoters of PIP and SWEET genes. Specifically, interactions were observed for three MYBs (MYB-05.1144, MYB-08.1369, and MYB-15.2534) and CCA1-12.452. Notably, CCA1 consistently bound to the SWEET_07.2532, SWEET_08.2947, and PIP_08.1447 promoters, reinforcing its potential regulatory role (Fig. 6B). The dual-luciferase and Y1H assays produced different results for PIF. Although PIF increased the activities of the tested SWEET and PIP promoters in the dual-luciferase assay, direct binding was not detected in the Y1H assay. Additional assays showed that PIF enhanced the activities of the MYB_01.1734 and MYB_15.2534 promoters (Fig. S7), whereas no direct interactions with these promoters were detected by Y1H. This discrepancy may reflect indirect regulation mediated by endogenous transcription factors or cofactors in Nicotiana benthamiana, a requirement for plant-specific post-translational modifications, or differences between the plant and yeast experimental systems. Moreover, non-specific promoter activation in the transient assay cannot be excluded.

Based on the assays described above, we identified LHY/CCA1 as a key transcription factor that regulates the expression of MYB, SWEET, and aquaporin genes involved in turgor-driven leaf movement. We therefore examined whether the promoters of the above genes contain Evening Element (EE) motifs, the known binding site of LHY/CCA1. EE motifs were detected in the promoters of chr07.2532_SWEET, chr08.2947_SWEET, and chr15.2534_MYB, located at −832 to −824 bp, −1493 to −1485 bp, and at three positions within the chr15.2534_MYB promoter (−193 to −185 bp, −901 to −893 bp, and −1419 to −1412 bp), respectively (Table S2). We cloned promoter fragments containing these EE motifs and performed Y1H assays, confirming that LHY/CCA1 bound to these regions. Importantly, when the corresponding EE motifs were mutated, binding was abolished (Fig. 7A).

Fig. 7.

LHY/CCA1 directly binds to EE motifs in the promoters of SWEET and MYB genes, linking circadian regulation to light-dependent leaf movement in peanut. A Yeast one-hybrid (Y1H) assay confirming the direct binding of LHY/CCA1 to the Evening Element (EE) motifs in the chr07.2532_SWEET, chr08.2947_SWEET, and chr15.2534_MYB promoters. The symbols “+” and “−” indicate the presence or absence of interactions, respectively. The AbA concentration used in each Y1H assay is shown below the corresponding panel. B Electrophoretic mobility shift assays showing the direct binding of MBP-CCA1-2.374 to EE motifs in the Sweet-08.2947 and MYB-15.2534 promoters. Biotin-labeled wild-type EE probes or the corresponding mutant EE probes were incubated with recombinant MBP-CCA1-2.374 protein. C Proposed working model linking circadian and light-responsive transcriptional regulation to candidate SWEET and aquaporin genes during peanut leaf movement. Solid lines represent experimentally supported transcriptional relationships, whereas dashed lines indicate proposed functional relationships that require further validation.

To further validate the direct binding of CCA1 to the EE motifs in these promoters, we performed electrophoretic mobility shift assays (EMSAs) using recombinant MBP-CCA1-2.374 protein. Clear shifted bands were observed when MBP-CCA1-2.374 was incubated with wild-type EE-containing probes from the pSweet-08.2947 and pMYB-15.2534 promoters, whereas no shift was detected in the absence of the protein. By contrast, mutating the EE motif strongly reduced or abolished the shifted signals. Moreover, the addition of a 100-fold excess of unlabeled wild-type competitor probe substantially weakened the shifted bands. These results demonstrate that CCA1 directly and specifically binds to the EE motifs in the pSweet-08.2947 and pMYB-15.2534 promoters in vitro (Fig. 7B).

These findings support transcriptional regulatory relationships between circadian/light-responsive transcription factors and the tested MYB, SWEET, and aquaporin genes. This network may contribute to coordinated sugar- and water-transport processes associated with turgor-driven leaf movement (Fig. 7C), a concept that requires direct functional validation.

3. Discussion

3.1. Light and circadian clock genes drive the rhythmic movement of leaves

Nyctinastic leaf movement is regulated by both external light cues and an internal circadian clock. Photoreceptors such as PHY and CRY detect day–night signals and help entrain the clock, which maintains rhythmic leaf motion even under constant light. Our transcriptome data revealed the strong expression of genes encoding these photoreceptors. Red light promotes leaflet closure by modulating phytochrome activity, whereas far-red light delays this process [23]. Arabidopsis cry mutants display altered circadian rhythms, confirming the roles of these photoreceptors in clock entrainment [24]. Our finding that the expression of circadian clock-related genes in peanut leaflets continued to cycle under continuous-light conditions further supports the presence of an internal oscillator driving nyctinasty.

This oscillator is built on conserved clock genes. We identified peanut homologs of LHY/CCA1, TOC1, and PRRs, whose expression patterns oscillated over the light/dark period. These genes mirror the Arabidopsis clock network, where LHY/CCA1 activate morning programs and repress evening genes like TOC1. In Arabidopsis and legumes, mutations in these genes disrupt the rhythms of leaf movement [9,25]. Similarly, in peanut, AhLHY/CCA1 expression peaked at dawn, while AhTOC1 expression peaked near dusk, indicating that a conserved light-clock system regulates rhythmic leaf movement.

3.2. Potential roles of SWEET and aquaporin genes in turgor-associated processes

While nyctinastic leaf movement is driven by changes in turgor in motor cells, these changes require the coordinated transport of water and solutes. Our study identified SWEET and aquaporin genes as candidate components associated with these processes. Their diurnal and light-responsive expression patterns are consistent with, but do not demonstrate, roles in regulating turgor.

Aquaporins are water channel proteins that control water flow across membranes. In nyctinastic plants such as Samanea saman, aquaporins are more active during leaf movement phases, helping motor cells quickly adjust their volumes [18]. Similar rhythmic expression has been observed in tobacco, which exhibits epinastic (growth-driven) leaf movements at dawn. The aquaporin NtAQP1, a PIP1 family member, was shown to be crucial for normal leaf unfurling; transgenic tobacco lacking NtAQP1 showed delayed or impaired leaf opening in the morning [16]. These examples illustrate a general principle: controlling water flux through aquaporin channels is a key mechanism by which plants affect organ movement. Here, we observed strong day/night changes in the expression of peanut AhPIP genes, with higher expression during the day coinciding with leaflet opening. This expression pattern is consistent with a possible contribution of these aquaporins to rhythmic water transport; however, their direct roles in peanut leaflet movement remain to be functionally tested.

Several SWEET genes also showed diurnal expression patterns in peanut leaves. Because SWEET proteins function as sugar transporters, their rhythmic expression may influence local sugar distribution and osmotic conditions. However, we did not directly measure sugar flux between extensor and flexor motor cells. Y1H and dual-luciferase assays supported transcriptional links between clock-related transcription factors and selected SWEET and aquaporin genes. These results identify SWEETs and aquaporins as candidate downstream components of the light and circadian regulatory network, but their contributions to turgor-driven leaflet movement require direct physiological and genetic validation.

These results, together with recent evidence for pulvinus-expressed ion channel genes in peanut [26], suggest that ion, sugar, and water transport are coordinately associated with nyctinastic movement. Our study extends the proposed pulvinus osmotic-motor model by identifying candidate light- and clock-responsive SWEET and aquaporin genes. However, whether these genes directly contribute to the cyclical swelling and shrinking of peanut motor cells remains to be determined through physiological and genetic analyses.

4. Materials and methods

4.1. Plant materials and treatment

Peanut seeds of uniform size and healthy appearance were selected from two genotypes: ZZ173 (spreading-type) and WF190 (erect-type). The seeds were surface-sterilized with 10% sodium hypochlorite for 15 min, rinsed five times with sterile water, and sown in pots filled with substrate. The plants were grown in a glasshouse at 25 °C with 60% relative humidity. At 30 days after sowing, treatment was initiated. Half of the seedlings were maintained under a 16 h light/8 h dark cycle (lights on at 5:30, off at 21:30) and designated as SN (spreading-type normal treatment) and EN (erect-type normal treatment). The other half were subjected to continuous 24 h light treatment, labeled as ST (spreading-type continuous light treatment) and ET (erect-type continuous light treatment). After 7 days, leaf samples were collected at 14 time points: 1 - 5:30, 2 - 6:00, 3 - 6:30, 4 - 7:00, 5 - 7:30, 6 - 10:30, 7 - 14:30, 8 - 18:30, 9 - 21:30, 10 - 22:00, 11 - 22:30, 12 - 23:00, 13 - 23:30, 14 - 2:30. The samples were sent to a sequencing facility (Novogene Co., Ltd. Tianjin, China) for transcriptome (RNA-Seq) analysis.

4.2. Measurement of photosynthetic parameters in peanut leaves

Photosynthetic parameters in peanut leaves were measured using a LI-6800P portable photosynthesis system (LI-COR Biosciences, USA). For each time point and treatment group, two leaves were measured from each of three individual plants, resulting in six biological replicates per time point per treatment. Measurements were taken hourly from 8:00 to 17:00, covering the full daylight period, to assess diurnal variation in photosynthetic activity. The parameters recorded included net photosynthetic rate (Pn, μmol m⁻² s⁻¹); stomatal conductance (Gs, mol m⁻² s⁻¹); intercellular CO₂ concentration (Ci, μmol mol⁻¹), and transpiration rate (Tr, mmol H₂O m⁻² s⁻¹). This setup allowed us to perform a detailed comparison of photosynthetic responses between light/dark cycles and continuous-light treatment conditions.

4.3. Transcriptome analysis

The raw transcriptome sequencing data underwent quality control, read alignment, transcript assembly, and quantification of gene expression. Differential expression and functional enrichment analyses were subsequently performed to identify differentially expressed genes and their associated biological functions. In addition, time-series expression analysis and co-expression network analysis were conducted. The overall workflow followed the method described by Cui et al. [27]. For time-series expression analysis, ClusterGVis was used, and genes with an FPKM value > 20 in at least one sample were retained for co-expression analysis [28]. Gene identifiers were assigned according to the nomenclature of the published telomere-to-telomere peanut reference genome [29]. Because cultivated peanut is an allotetraploid species containing A and B subgenomes, chromosome-based identifiers were retained to distinguish homeologous genes located on different chromosomes. To facilitate cross-referencing with previous studies, the genes analyzed in this study were mapped to the corresponding gene models in the Tifrunner reference genome. A complete correspondence between the chromosome-based identifiers, T2T genome locus IDs, Tifrunner gene IDs, and functional annotations is provided in Table S3. Under this naming system, the chromosome number and the ordinal position of the annotated gene on that chromosome are included in the identifier. For example, chr06.3134_ELF3 represents the 3,134th annotated gene on chromosome 6, which was functionally annotated as ELF3.

4.4. RT-qPCR validation of the transcriptome data

To validate the reliability of the transcriptome data, 11 light- and circadian clock-related genes were selected for RT-qPCR based on their differential expression in the transcriptome data. The experimental procedures and reference gene used were described by Cui et al. [27]. The primer sequences used for RT-qPCR are listed in Table S4.

4.5. Yeast one-hybrid (Y1H) assay

Y1H assays were performed using the Matchmaker Gold Yeast One-Hybrid Library Screening System (Clontech, Cat. No. 630491). Briefly, approximately 2000 bp of the promoter sequence of the target gene was cloned into the pAbAi vector. The recombinant vector was linearized and used to transform yeast cells, followed by self-activation testing to determine the appropriate Aureobasidin A (AbA) concentration for selection. The coding sequences of transcription factor genes were cloned into the pGADT7 vector to generate AD constructs. These constructs were subsequently used to transform the corresponding yeast strains, and protein–DNA interactions were assessed based on growth on SD/-Leu medium supplemented with the predetermined concentration of AbA. The primers used for promoter and TF gene cloning are listed in Table S3.

4.6. Dual-luciferase assay

Approximately 2000 bp of the promoter sequence of each target gene was cloned into the pGreen II 0800-LUC vector. The coding sequence of the transcription factor gene was inserted into the pGreen II 0029 62-SK (SK) vector. The resulting constructs were used to co-transform Agrobacterium tumefaciens strain GV3101 cells (carrying the p19 silencing suppressor), which were then co-infiltrated into Nicotiana benthamiana leaves. After 48 h, luciferase activity was visualized using an in vivo imaging system (NightShade LB985, Berthold, Germany), and the LUC/REN ratio was quantified using a Dual-Luciferase Reporter Assay kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. The primers used for construct generation are listed in Table S3.

4.7. Electrophoretic mobility shift assay

Electrophoretic mobility shift assays (EMSAs) were performed to examine the binding of CCA1 to EE motifs in the promoters of Sweet-08.2947 and MYB-15.2534. The coding sequence corresponding to CCA1-2.374 was cloned into an MBP-fusion expression vector and expressed in Escherichia coli. The recombinant MBP-CCA1-2.374 protein was purified using an MBP-tagged Protein Purification kit according to the manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China). Biotin-labeled oligonucleotide probes containing the wild-type EE motifs, the corresponding mutant probes, and unlabeled competitor probes were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). Complementary oligonucleotides were annealed to generate double-stranded probes. For each binding reaction, the biotin-labeled probe was incubated with purified MBP-CCA1-2.374 protein in EMSA binding buffer at room temperature. For competition assays, a 100-fold molar excess of unlabeled wild-type probe was added to the reaction before adding the labeled probe. The DNA–protein complexes were separated on a native polyacrylamide gel and transferred to a nylon membrane. Signals were detected using a Chemiluminescent EMSA Detection kit according to the manufacturer’s protocol (Beyotime Biotechnology, Shanghai, China). Binding specificity was evaluated by comparing the mobility shifts of wild-type probes, mutant probes, and reactions containing unlabeled competitor probes. The primers are listed in Table S3.

4.8. Statistical analysis

All statistical analyses were conducted using GraphPad Prism software. For comparisons between two groups, an unpaired Student’s t-test was used to evaluate differences at the same time point. When more than two groups were compared, one-way ANOVA followed by the appropriate post hoc tests was performed to determine statistical significance across treatments at each time point. Significance levels are indicated as P ≤ 0.05 (∗) and P ≤ 0.01 (∗∗) in the figures.

CRediT authorship contribution statement

Yuanyuan Cui: Writing – review & editing, Writing – original draft, Visualization, Validation, Investigation, Formal analysis, Data curation. Haosong Guo: Validation, Investigation, Formal analysis, Data curation. Xiaoyu Liu: Validation, Investigation, Data curation. Shuai Ding: Formal analysis, Data curation. Xiaoqin Liu: Writing – review & editing, Resources, Funding acquisition, Data curation.

Declaration of competing interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

The transcriptome data were deposited in the NCBI Sequence Read Archive (BioProject: PRJNA1243696).