Bioorganic studies on the nyctinastic leaf-movement of plants

Abstract

Nyctinasty, a circadian-regulated leaf movement observed in legumes, has long intrigued scientists. This study identified genus-specific endogenous chemical factors, leaf-closing factors (LCFs) and leaf-opening factors (LOFs), that control this rhythmic behavior. In Samanea saman, a model for nyctinasty, the LCF 12-hydroxyjasmonic acid glucoside induces leaf-folding by selectively targeting extensor motor cells and activating reactive oxygen species (ROS)-dependent K⁺ efflux via the SPORK2 channel. Functional analyses revealed that SPORK2 and the SsSLAH1/SsSLAH3 anion channel complex are key effectors in the regulation of leaf movement. Remarkably, SPORK2 also functions as a temperature-sensitive K⁺ channel, inactivating at low temperatures to mediate rain-induced leaf folding, a phenomenon underlying the nickname “rain tree”. These findings provide a chemical entry point to dissect the molecular machinery of nyctinasty and reveal a previously unrecognized role for plant K⁺ channels in temperature sensing.

Chemical factors inducing leaf-movement of Samanea saman

1. Introduction

Nyctinasty is a circadian-regulated rhythmic leaf movement, most famously observed in leguminous plants such as Mimosa pudica, Samanea saman (“sleeping tree” or “rain tree”) (Fig. 1A), Albizia julibrissin (“silk tree”), and various other genera within the family Fabaceae.^1)–14)) In such species, the leaves or leaflets fold in the evening and reopen at dawn (Fig. 1A), tracking an internal biological clock even under continuous light or continuous darkness.^15),16)) Because this phenomenon is so conspicuous and reliably follows a circadian timetable, it has historically attracted the attention of scientists. Paleobotanical evidence suggests that foliar nyctinasty may have originated as early as 250 million years ago.¹⁷⁾⁾ The discovery of fossilized leaves exhibiting bilaterally symmetrical feeding traces, a pattern consistent with rhythmic leaf folding, indicates that nyctinastic movements were already established in ancient seed plants. These fossils are attributed to gigantopterids, an extinct lineage predating the rise of angiosperms by over 100 million years. This temporal gap strongly suggests that the evolutionary origin of “sleep movements” is deeply rooted in the history of vascular plants. The emergence of foliar nyctinasty likely represented a pivotal adaptation in the early diversification of land plants, especially within the lineage that would later give rise to modern legumes.

Figure 1.

Nyctinastic leaf movement of Samanea saman. (A) S. saman exhibits rhythmic leaf folding at dusk and unfolding at dawn. This movement is under circadian control and regulated by the plant endogenous biological clock. (B) Nyctinastic movement is mediated by a specialized motor organ, the pulvinus, located at the base of the leaf. Coordinated volume changes in the abaxial (flexor) and adaxial (extensor) pulvini drive the opening and closing of the leaves. (Curr. Biol., 28, 2230–2238.e7.¹⁶⁾⁾)

The earliest record on nyctinasty dates back to the era of Alexander the Great (4th century BCE): Androsthenes of Thasos (admiral to Alexander the Great) documented nightly leaf-folding in a tamarind-like plant.¹⁸⁾⁾ In 1729, the French astronomer Jean-Jacques d’Ortous de Mairan provided experimental evidence that Mimosa pudica continued its leaf-folding rhythm in total darkness, a pivotal observation that sparked modern investigations of endogenous biological clocks.¹⁹⁾⁾ Charles Darwin and his son Francis conducted meticulous work on plant movements in the 19th century, summarized as a book ‘The Power of Movement in Plants’ (1882).²⁰⁾⁾ Their studies included some of the earliest systematic analyses of nyctinasty. They not only tracked the diurnal and nocturnal movements of a variety of species but also recognized that leaf-folding was not merely a mechanical response to external light but rather a self-sustained oscillation within the plant.

Subsequent studies demonstrated that pulvinar motor cells drive the leaf-folding and leaf-opening movements through volume changes controlled by osmotic gradients, ion fluxes, and water transport (Fig. 1B).^{4),9),21)–23))} These cellular events, in turn, are programmed by internal circadian clocks that coordinate the daily timing of ion-channel expression and activity. Furthermore, compelling evidence has emerged that plants produce endogenous chemical factors (leaf-closing substances, leaf-opening substances) that directly elicit or inhibit leaf-folding.

The present review aims to integrate historical perspectives on nyctinasty with modern advances in physiology, molecular genetics, and, in particular, natural products chemistry. This review article covers chemical aspects of nyctinasty, including the discovery of endogenous chemical factors that induce or prevent leaf movement with their mode of action, the biological aspects of nyctinasty including the identification of molecular players, including their role in the regulation of leaf-movement, and the link between chemistry and biology.

2. Nyctinasty as the “Rosetta Stone” analogy for the circadian clock

In the early to mid-20th century, Erwin Bünning hypothesized that an endogenous circadian oscillator underpins nyctinasty, along with other rhythmic phenomena such as flowering time. By working extensively with Phaseolus beans, Bünning established many cornerstones of circadian biology in plants. Indeed, nyctinasty was sometimes likened to a “Rosetta Stone” for the circadian clock, implying that deciphering the mechanism of rhythmic leaf-folding might illuminate the entire clock network.¹⁾⁾

In the 1960s to 1980s, numerous physiological experiments were performed, notably by Ruth Satter and colleagues, who used the large tropical legume Samanea saman as a model.^{1),15),22)–24))} They clarified the general principle that nyctinasty depends on reversible changes in turgor pressure in specialized motor cells in the pulvinus (the swollen petiole base) (Fig. 1B).^22),25),26)) Turgor depends on ion flux and water transport, reminiscent of the guard cell mechanism controlling stomatal aperture.⁹⁾⁾ These achievements disclosed how the pulvinus accomplishes leaf movements. A pulvinus typically appears as a bulbous, flexible organ at the leaf or leaflet base. It contains two opposing regions: extensor (adaxial or upper) motor cells and flexor (abaxial or lower) motor cells (Fig. 1B). During the daytime, these cells typically display an “anti-phase” relationship in volume: if the flexor cells lose volume (shrink), the extensor cells may swell, and vice versa. This difference in turgor pressure on opposite sides of the pulvinus causes the entire leaf (or leaflet) to rotate or fold. Pulvinar motor cells accumulate or release ions (particularly K⁺ and Cl⁻) through ion channels in the plasma membrane. Because water follows osmotic gradients, changes in ionic concentration drive water flux across the membrane, altering cell volume. The net effect is swelling or shrinking of specific motor cells.

Tremendous efforts have been made to discover candidate genes encoding putative potassium channel, aquaporins, etc., from S. saman (Table 1).^27)–29)) However, elucidating the precise molecular factors, channels, signaling pathways, and regulatory genes, proved difficult. S. saman is a non-model plant that has fallen outside the current trends of genome biology, and as of 2025, no genetically modified Samanea plants have been successfully prepared, making it difficult to analyze the underlying gene network that regulates nyctinasty. Thus, it seems that no incident to mirror the appearance of “Champollion” in the “Rosetta Stone” story ever occurred.

Table 1.

List of K⁺ and Cl⁻ channels identified in Samanea saman

Name	Function	Sequence identity (%)	Sequence homologya (%)
SPORK1	Putative K+out channel	-b	-b
SPORK2	K+out channel	96.4b	96.9b
SPORK3	Putative K+out channel	47.0b	58.4b
SPORK4	Putative K+out channel	49.8b	64.5b
SPICK1	Putative K+in channel	-c	-c
SPICK2	Putative K+in channel	64.7c	78.3c
SPICK3	Putative K+in channel	37.1c	54.8c
SPICK4	Putative K+in channel	35.2c	52.9c
SsSLAH1	Anion channel	-d	-d
SsSLAH3	Anion channel	35.8d	56.1d

^aThe values were calculated using the BLOSUM62 substitution matrix.

^bThe values were calculated by pairwise alignment against the SPORK1 protein sequence.

^cThe values were calculated by pairwise alignment against the SPICK1 protein sequence.

^dThe values were calculated by pairwise alignment against the SsSLAH1 protein sequence.

3. Identification of endogenous chemical factors controlling nyctinasty

Alongside physiological approaches, researchers noted in early studies that aqueous extracts from the pulvinus of Mimosa pudica appeared to contain a transferrable “factor” capable of inducing leaf folding.³⁰⁾⁾ Some decades later, a putative “plant hormone” named turgorin was reported,¹⁸⁾⁾ proposed to be the long-sought endogenous inducer of leaf-folding movement. The isolation of turgorin was guided by its ability to induce rapid leaf-folding in Mimosa, and subsequent studies demonstrated that the compound exhibits bioactivity across a wide range of plant species. However, subsequent work showed that the initially reported structure was likely an artifact: strongly acidic partial structures turned out to be chemically implausible in planta, and a purified salt form lacked activity.³¹⁾⁾ In addition, although foliar nyctinasty is classified as a type of tropic movement, its underlying mechanism is fundamentally distinct from the rapid thigmonastic movement observed in Mimosa pudica.^32)–34)) Nyctinastic leaf movements are driven by circadian-controlled ion fluxes and water redistribution, proceeding on a much slower timescale. Therefore, using thigmonastic response of Mimosa as a model system for identifying endogenous bioactive compounds involved in nyctinasty may be misleading. Yamamura et al. isolated several leaf motility factors that functioned as potassium salts.^31),35),36)) However, it was debatable whether these were genuine endogenous bioactive factors. This confusion underscored the difficulty in combining classical extraction/fractionation with robust bioassays for leaf-folding. In these early studies, active substances were isolated based on their ability to induce movement in the leaves of plants such as Mimosa plant, which is both easy to use and highly sensitive. This was because it was initially assumed that the endogenous substances responsible for leaf-movement were novel plant hormones ubiquitous in all plants. As in previous experiments, researchers may be misled when evaluating an extract from one species of plant in a bioassay with another species, or when artefacts are introduced inadvertently during the extraction and isolation processes.

Ueda et al. overcame such pitfalls and succeeded in identifying endogenous chemical factors controlling nyctinastic leaf movements.^7),8),37)) They employed a consistent protocol as follows: leaves (including petioles/pulvini) from one species, typically at a known time of day, were excised and extracted. The purification of chemical factors was guided by a bioassay monitoring biological activity using fresh excised leaflets of the same species, ensuring that the observed bioactivity was relevant to natural chemical factors of that species (Fig. 2A). Applying this approach to multiple legume genera (Phyllanthus, Lespedeza, Cassia, and Samanea, etc.) revealed that many species contain two distinct classes of compounds (Fig. 2B): Leaf-closing factors (LCFs, 1–5 in Fig. 2B) inducing leaflet folding even under conditions that normally keep leaves open and Leaf-opening factors (LOFs, 6–10 in Fig. 2B) promoting leaflet opening and overriding or delaying the folding process at night (Fig. 2A).^{8),37)–49))} In a bioassay where detached leaves absorbed aqueous solutions of the active compounds, LCFs exhibited biological activity at concentrations on the order of 10⁻⁶ to 10⁻⁷ mol/L, whereas LOFs showed activity at concentrations ranging from 10⁻⁵ to 10⁻⁶ mol/L. Notably, these two activities appear to antagonize each other, complicating fractionation if the two classes co-elute at early purification stages. One of the most striking conclusions was the structural diversity of LCFs and LOFs among different leguminous plants (Fig. 2).⁵⁰⁾⁾ Another point to consider is the genus-specific biological activity of each LOF and LCF. LOFs and LCFs are only effective on leaves from the same genus.^50)–52)) Rather than one universal plant hormone, each genus often had a unique pair of compounds. Some well-characterized examples include:

Figure 2.

Leaf-closing factor (LCF) and leaf-opening factor (LOF) in leguminous plants. (A) Bioassay system used to evaluate the biological activities of LCFs and LOFs. Application of phyllanthurinolactone (1, 1 × 10⁻⁷ mol/L) as LCF induced leaf closure of Phyllanthus urinaria L. (left), whereas potassium isolespedezate (8, 1 × 10⁻⁶ mol/L) as an LOF inhibited leaf closure of Cassia mimosoides L (right). The red arrows indicate the leaflets treated with 1 or 8, respectively. All other leaflets were treated with water (mock). (B) Chemical structures of five pairs of LCFs and LOFs identified from P. urinaria, S. saman, Albizia julibrissin, Lespedeza cuneata, C. mimosoides, and Mimosa pudica.

Phyllanthurinolactone (1), an LCF of Phyllanthus urinaria.³⁹⁾⁾ A lactonic compound shown to induce leaf folding (“closing factor”). Notably, the aglycone of phyllanthurinolactone is inactive, with the active glycoside form building up in the evening. This daily oscillation in free vs. conjugated forms aligns with the plants’ typical onset of leaf-folding behavior at dusk.

12-Hydroxyjasmonic acid glucoside (2), an LCF of Samanea saman and the closely related genera Albizia julibrissin.^47)),53)) Particularly significant was the discovery that in S. saman, 12-hydroxyjasmonic acid glucoside (2, JAG), a glucoside of 12-hydroxyjasmonic acid, can strongly induce leaf folding. Surprisingly, although 2 is structurally related to jasmonic acid (JA), a well-known plant hormone in defense, wound response, and senescence, JAG does not appear to induce canonical JA-related responses such as the expression of LOX2 or OPCL1 genes or the production of volatile organic compounds.⁵³⁾⁾ Instead, it shows a highly specific effect on nyctinastic folding. Free acid JA, JA–Ile, or coronatine (a JA-Ile mimic) fail to cause leaf movements. Thus, JAG occupies a distinct functional niche from the canonical JA pathways.

p-Coumaroylagmatine (7), an LOF of Samanea saman and the closely related genera Albizia julibrissin.⁴⁰⁾⁾ Some species produce coumaroyl conjugates of agmatine that act as opening factors. They can override the leaf-folding process even during subjective nighttime if applied at sufficient concentrations.

Chemically, LCFs and LOFs encompass a diverse array of structures, including lactones, jasmonate derivatives, and alkaloid-like compounds. Despite their structural diversity, they share a common feature, inducing leaf-folding or leaf-opening movement in a genus-specific manner. This chemical perspective provided a crucial link between plant physiology and molecular biology. The finding that certain compounds may specifically induce or inhibit leaf movements, even in excised pulvinus preparations, led to a pivotal question: how do LCFs and LOFs precisely trigger volume changes in motor cells? This marked the beginning of a more chemistry-driven approach to elucidating the molecular mechanisms underlying nyctinastic movement.

4. Enzymatic hydrolysis of glucoside regulating the balance between LCF and LOF

Analyses of plant extracts over the 24-hour cycle revealed that the ratio of LCF to LOF changes dramatically in nyctinastic species. For example, in Phyllanthus urinaria, the active LCF phyllanthurinolactone (1) accumulates at dusk to about 20-fold its daytime level (Fig. 3).⁵⁴⁾⁾ This rhythmic fluctuation stems from enzymatic hydrolysis of phyllanthurinolactone to its corresponding aglycone. A similar case can be found in glycoside-type LOF of Lespedeza cuneata (8).^55)–57)) According to this hydrolysis, the β-glucosidase activity of the plant extract changes diurnally. However, the glucosidase responsible for 1 remains unidentified, because the source plant, P. urinaria, is a non-model species. In addition, for the glucoside-type LOF, potassium isolespedezate (8), the levels of which fluctuate diurnally,⁵⁸⁾⁾ we reported that glycosidase activity in crude enzyme extracts prepared from the source plant also varied diurnally. Moreover, partial sequences of glycosidase associated with 8 were obtained through affinity purification; however, complete cloning has not yet been accomplished.⁵⁹⁾⁾ Thus, it is hypothesized that enzymes such as glycosyltransferases or glucosidases can add or remove sugar moieties, thereby altering the bioactivity of LCF/LOF.

Figure 3.

Rhythmic fluctuation in endogenous levels of LCF and LOF in Phyllanthus urinaria. Circadian leaf movement in P. urinaria correlates with rhythmic changes in the endogenous balance between LCF and LOF.

5. Why do plants fold their leaves?

The physiological significance of nyctinastic leaf movements has intrigued scientists for over a century. Charles Darwin suggested that this behavior protects leaves from chilling or freezing,²⁰⁾⁾ whereas Erwin Bünning proposed that it prevents moonlight interference with the photoperiodic clock.⁶⁰⁾⁾ However, despite these long-standing hypotheses, no direct experimental evidence had been available to support them.

A major barrier to such investigations has been the absence of molecular tools capable of selectively inhibiting nyctinastic movement. Molecular genetic studies employing gene knockout lines have proven to be powerful tools in dissecting plant signal transduction pathways. Nevertheless, mutant lines specifically associated with nyctinastic leaf movement remain exceedingly rare. To date, only a few mutants affecting tissue differentiation of the pulvinus, ones lacking the motor organ, have been described,^61)–64)) but no mutants directly implicated in the signal transduction of nyctinasty have been identified.

Chemical identification of LOF and LCF have overcome this limitation, leading to the development of artificial leaf-movement factors that can induce a state of persistent leaf opening, or “insomnia”, in plants. These compounds were designed based on the known mechanism of nyctinasty, where β-glucosidase converts an active glycoside into an inactive aglycone. Modifying the structure to resist hydrolysis led to the synthesis of stable analogues that prevent leaf-folding by evading enzymatic deactivation.^65)–67))

Among these compounds, potassium isolespedezate (8 in Fig. 2B) and its l-glucoside analogue (ent-8) were particularly effective (Fig. 4).⁶⁶⁾⁾ We demonstrated that the enantiomeric l-glucoside (ent-8) was as effective as the d-glucoside (8). In addition, we synthesized β-d-galactoside and α-l-mannoside analogues and demonstrated that both of these sugar derivatives were as effective as 8.⁶⁸⁾⁾ Thus, we concluded that the structure of the sugar moiety in 8 does not affect its leaf-opening activity, and we examined the effect of continuous leaf-opening using ent-8. Although compound 8 induced transient leaf opening for about 2 days until enzymatic hydrolysis removed it, compound ent-8 maintained leaf-opening activity for over a week. Leaves treated with ent-8 failed to close at night, eventually withering and dying (Fig. 4). This “insomnia” phenotype provided the first experimental demonstration that nyctinastic leaf-folding is essential for plant survival. These results finally offer an experimental answer to the question posed by Darwin and Bünning: “Why do leguminous plants sleep?”

Figure 4.

Enantiomeric LOF resistant to enzymatic hydrolysis induces “insomnia” in the leaflet of Cassia mimosoides. Chemically synthesized enantiomeric LOF (ent-8, 3 × 10⁻⁶ mol/L), which resists enzymatic hydrolysis in planta, persistently inhibits leaf closure in C. mimosoides, resulting in sustained leaf opening (“insomnia”). Leaf opening of Cassia leaflets treated with ent-8 at 19:00 (2 hours after light-off). From left to right: 2 days after treatment, 4 days after treatment, 9 days after treatment, and a 9-day untreated control (mock). (Tetrahedron Lett., 43, 7545–7548.⁶⁵⁾⁾)

6. Chemical biology approach to the mode of action of nyctinasty

Although numerous LCFs and LOFs have been identified, only a few have been examined in detail at the mechanistic level. Ueda and coworkers pioneered the use of molecular probes, such as fluorescently labeled and photoaffinity-labeled analogs of LCFs and LOFs, to investigate their modes of action through a chemical biology approach.^{51),69)–73))} A notable achievement of this work was obtained using JAG (2), an LCF identified in S. saman.

When using molecular probes to investigate the in vivo mechanisms of a bioactive compound, one of the most serious issues is non-specific binding of the probe to biological tissues. In their early studies, Ueda and coworkers encountered substantial difficulty with non-specific binding of probes in plant tissues. However, they overcame this issue by developing and employing a unique enantio-differential strategy (Fig. 5).^74)–76)) This strategy involves labeling both the natural enantiomer and its unnatural enantiomer with a fluorophore and comparing their binding patterns within living tissues. Although the two enantiomers share identical physicochemical properties, which are the main cause of non-specific binding, only the natural enantiomer exhibits bioactivity that induces leaf folding, implying differential affinity for the receptor. This comparison enables the effective elimination of the effect of non-specific binding.

Figure 5.

Enantioselective fluorescence imaging showing the target cell of LCF in Samanea saman. (A) Structures of natural-type fluorescence probe (11) and enantiomer-type fluorescence probe (12). (B) These images show cross-sections of the leaf pulvini cut perpendicular to the vascular bundle. The vascular bundle is characteristically curved in a “C” shape, and its ventral side corresponds to the adaxial (extensor) side of the leaf face. Fluorescent imaging experiments demonstrated that only the natural-type fluorescence probe (11) selectively binds to extensor motor cells in the pulvini of young S. saman, whereas nonspecific tissue binding was observed for both enantiomers. When an excess amount of JAG (2) was added, fluorescence of 11 on the extensor side disappeared, but fluorescence of 11 in the vascular tissue did not.⁷⁵⁾⁾ In the primary root and hypocotyl no differences were detected between the fluorescence images obtained after treatment with probe 11 or 12, suggesting that only nonspecific binding had occurred.⁷⁵⁾⁾ (Angew. Chem. Int. Ed. Engl., 47, 7289–7292.⁷⁴⁾⁾)

Ueda et al. showed that the enantiomer of 2 lacked leaf-folding activity and additionally revealed that the structure of the sugar moiety did not affect the leaf-folding activity.^74),77)) The molecular designs of the following fluorescence/photoaffinity probes are based on these results. Using both enantiomers of fluorescently labeled 2 (11 and 12), Ueda et al. demonstrated that the natural enantiomer preferentially bound to the extensor motor cells on the adaxial (upper) side of the pulvinus (Fig. 5).⁷⁴⁾⁾ They further applied a similar strategy to develop a photoaffinity probe by introducing a photoreactive diazirine group into the sugar moiety of 2 (Fig. 6), while maintaining its leaf-closing activity (13 and 14). Photoaffinity labeling was then performed using live protoplast of extensor motor cells. Comparison of crosslinking profiles of the natural and mirror-image enantiomers (and subtracting background signals) revealed that JAG binds to an unidentified protein in the plasma membrane fraction from extensor motor cells in a stereospecific manner (Fig. 5).⁷⁴⁾⁾

Figure 6.

Enantiodifferential photoaffinity labeling of the target protein of LCF in Samanea saman. (A) Structures of natural-type photoaffinity probe (13) and enantiomer-type photoaffinity probe (14). (B) Photoaffinity-labelling experiments were performed on the plasma membrane fraction prepared from protoplasts of collected extensor motor cells. The protoplasts were prepared from the extensor pulvini excised from S. saman leaves. The results demonstrated that the natural-type photoaffinity probe (13) specifically binds to a membrane protein in protoplasts of living motor cells in S. saman. Selective labeling of a 38 kDa smear band was observed only with 13, and this band disappeared in the presence of an excess amount of JAG (2). (Angew. Chem. Int. Ed. Engl., 47, 7289–7292.⁷⁴⁾⁾)

Thus, 2 induces nyctinastic leaf-folding by specifically targeting the upper extensor motor cells of the pulvinus, causing their shrinkage and thereby pulling the leaflet closed.

7. Physiology and molecular biology of plant nyctinasty

Understanding of biological phenomena such as nyctinasty requires not only a chemical but also a molecular biological approach. Next, we will introduce the process by which molecular biological studies have elucidated the molecular players that regulate plant leaf-movement.

7.1. Pioneering work by Nava Moran and coworkers.

Volume changes in motor cells that enable nyctinastic movements require substantial transmembrane fluxes of K⁺, Cl⁻, and water.²⁾⁾ This suggested the presence of specific transporter proteins mediating K⁺/Cl⁻/H₂O transport in these cells. Following the development of the patch-clamp technique as a revolutionary method for measuring cellular ion channel activity,⁷⁸⁾⁾ Nava Moran and colleagues applied this approach to demonstrate for the first time the presence of outward-rectifying K⁺ (K⁺_out) channels in Samanea motor cells.⁷⁹⁾⁾ In 2001, Moran’s group also reported the existence of hyperpolarization-activated, inward-rectifying K⁺ (K⁺_in) currents in Samanea motor cells.⁸⁰⁾⁾

K⁺ channel genes were cloned from plants.^81),82)) In the model plant Arabidopsis thaliana, nine Shaker-like genes were isolated: seven K⁺_in genes comprising K⁺ channel in Arabidopsis thaliana 1 (KAT1)/KAT2/KAT3 and Arabidopsis K⁺ transporter 1 (AKT1)/AKT2/AKT5/AKT6, and two K⁺_out genes, Stelar K⁺ Outward Rectifier (SKOR) and Guard cell Outward Rectifying K⁺ channel (GORK).⁸³⁾⁾ Using a cDNA library constructed from Samanea motor cells, Moran and coworkers cloned a K⁺_out gene SPORK1 (for Samanea Pulvini Outward-Rectifying K channel no. 1), as an ortholog of SKOR/GORK, and the K⁺_in genes SPICK1 and SPICK2 (for Samanea Pulvini Inward-rectifying Channel for K⁺ no. 1 and no. 2, respectively), as orthologs of AKT2.²⁹⁾⁾ These three Samanea K⁺ channel candidates contained the conserved TXGYG motif, the universal K⁺ selectivity filter sequence,⁸⁴⁾⁾ strongly suggesting their involvement in transmembrane K⁺ transport. Unfortunately, these genes were not able to be functionally reconstituted in common heterologous expression systems including Xenopus laevis oocytes, Sf9 insect cells, and mammalian HEK-293T cells, thus preventing definitive proof of their function as K⁺ channels mediating nyctinastic movements in motor cells. Thus K⁺ channel regulating nyctinasty remains unidentified.

Transmembrane water transport in biological systems is facilitated by aquaporin proteins that function as molecular water channels.⁸⁵⁾⁾ Thus, aquaporins were predicted to mediate the rapid water fluxes necessary for volume changes in motor cells.^2),86)) According to the identification of Arabidopsis aquaporin family genes,⁸⁷⁾⁾ Moran and coworkers cloned two aquaporin homologs from a Samanea pulvinar cDNA library: SsAQP1 and SsAQP2.²⁸⁾⁾ In the Xenopus oocyte heterologous expression system, SsAQP2 demonstrated high water transport capacity, and SsAQP1 exhibited relatively low water permeability. In contrast, gene expression analysis revealed that only SsAQP2 maintained rhythmic expression in constant darkness, suggesting circadian regulation. Moran and colleagues concluded that SsAQP2 plays a crucial role in the regulation of nyctinastic leaf movements.²⁸⁾⁾

7.2. Recent advances in understanding Samanea ion channels.

The putative K⁺ channels (SPORK1/SPICK1/SPICK2) identified by Moran et al. lacked definitive functional evidence, as their K⁺ transport activity was unable to be detected in heterologous expression systems.²⁹⁾⁾ In addition, recent studies demonstrated that Cl⁻ channels play an equally significant role as K⁺ channels in volume changes of plant cell; however, Cl⁻ channels in the motor cells of S. saman had remained uncharacterized.⁹⁾⁾

In 2008, two research groups independently reported that the SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) gene encodes a plasmalemma Cl⁻ channel in Arabidopsis guard cells.^88),89)) Moreover, four homologs of SLAC1, designated as SLAC1 HOMOLOGUE 1 (SLAH1)/SLAH2/SLAH3/SLAH4, were identified and shown to encode anion channel subunits.^90)–93)) During this period, next-generation sequencing technologies became widely available, accelerating the discovery of novel genes in non-model species.⁹⁴⁾⁾ According to the development of scientific and technological advancements in the mid-2010s, Ueda et al. conducted transcriptome analysis of cDNA libraries from Samanea motor cells using next-generation sequencing, successfully identifying a total of 71,473 genes.¹⁶⁾⁾ Among them, they identified two Cl⁻ channel gene candidates, SsSLAH1 and SsSLAH3, which are orthologs of SLAH1 and SLAH3, respectively (Fig. 7A). They also identified five novel K⁺ channel gene candidates. For K⁺_out genes, we isolated SPORK2/SPORK3/SPORK4 as homologs of SPORK1 and orthologs of SKOR/GORK. For K⁺_in genes, Ueda et al. identified SPICK3/SPICK4 as homologs of SPICK1/SPICK2 (Fig. 7B). Using a Xenopus oocytes expression system, Ueda et al. examined the transport activities of these newly identified Cl⁻ and K⁺ channel candidates. SsSLAH1 and SsSLAH3 showed no transport activity individually but exhibited Cl⁻ conductance when co-expressed, consistent with the behavior of SLAH1 and SLAH3 from Arabidopsis.⁹³⁾⁾ SPORK2 displayed slowly activating K⁺_out currents in response to depolarization, whereas SPORK3/SPORK4 and SPICK3/SPICK4 showed no detectable transport activity. Furthermore, whole-cell patch-clamp measurements of Samanea motor cells detected Cl⁻ currents and K⁺_out currents with characteristics similar to those of the SPORK2 and SsSLAH1-SsSLAH3 complex when expressed in Xenopus oocytes. These findings strongly suggested that SPORK2 functions as a K⁺_out channel and the SsSLAH1-SsSLAH3 complex as a Cl⁻ channel in Samanea motor cells.

Figure 7.

Molecular phylogeny of Cl⁻ and K⁺ channel genes in Samanea saman. (A and B) Unrooted neighbor-joining trees of Cl⁻ channels (A) and K⁺ channels (B) in S. saman and Arabidopsis thaliana. Samanea genes are colored in red and Arabidopsis genes in black. Note that the amino acid sequence of SPORK1 shows high similarity to SPORK2. The scale denotes substitutions per site.

7.3. Ion channel-mediated molecular mechanism of Samanea leaf-movement.

To elucidate the potential involvement of SPORK2 and SsSLAH1/SsSLAH3 in nyctinastic movements, Ueda et al. conducted temporal expression analyses of these genes.¹⁶⁾⁾ SPORK2 exhibited expression maxima in the morning in both flexor and extensor cells, whereas SsSLAH1 displayed morning-specific expression peaks exclusively in flexor cells. In contrast, SsSLAH3 showed constitutive expression without significant temporal variation. Notably, these expression rhythms persisted under both light-dark cycles and constant light conditions, suggesting endogenous regulation. Based on these differential expression patterns, they hypothesized that the SsSLAH1-SsSLAH3 complex may form specifically in flexor cells during the morning phase.

From these findings, Ueda et al. proposed the following molecular mechanism underlying nyctinastic leaf opening (Fig. 8): (1) In the morning, the SsSLAH1-SsSLAH3 complex forms specifically in flexor cells, mediating Cl⁻ efflux into the extracellular space and inducing membrane depolarization; (2) This depolarization activates SPORK2, facilitating K⁺ efflux; (3) The resulting KCl efflux drives osmotic water movement (likely through SsAQP2), leading to flexor cell shrinkage and consequent leaf opening. The critical aspect of this mechanism is the cell-specific expression of SsSLAH1, which occurs exclusively in flexor cells during the morning, despite the presence of SPORK2 and SsSLAH3 in both flexor and extensor cells. This spatiotemporal expression pattern of SsSLAH1 appears to function as a molecular ‘switch’ that specifically triggers leaf opening in Samanea.

Figure 8.

Possible molecular mechanism of nyctinastic leaf opening at dawn in Samanea saman. Schematic representation of the proposed mechanism for leaf opening. During leaf opening, SsSLAH1 expression is upregulated specifically in flexor motor cells, leading to SsSLAH3 activation and subsequent anion efflux. The resulting membrane depolarization activates SPORK2 channels, causing K⁺ efflux. These coordinated ion movements decrease cell volume in flexor motor cells relative to extensor cells, driving nyctinastic leaf opening.

To validate the functional significance of SsSLAH1, genetic disruption studies may be ideal; however, genetic transformation protocols have not been established for Samanea. To circumvent this limitation, Ueda et al. generated GmSLAH1 knockdown lines in Glycine max, a leguminous model plant phylogenetically related to Samanea.¹⁶⁾⁾ These transgenic plants exhibited significantly attenuated leaf folding kinetics, providing genetic evidence for the essential role of SLAH1-type anion channels in nyctinastic leaf movements.

8. Link between endogenous chemical factors and ion channels

The next problem is to unravel the missing links between chemistry and biology, such as between 2 and newly identified ion channels. Analyses by Ueda et al. revealed that 2 selectively targets extensor cells to promote reactive oxygen species (ROS) accumulation, which triggers cellular shrinking (Fig. 9A).⁹⁵⁾⁾ Plant hormones such as jasmonic acid and abscisic acid, which induce ROS accumulation in Arabidopsis guard cells, did not induce ROS accumulation in extensor cells. Conversely, 2 did not induce ROS accumulation in Arabidopsis guard cells. These results suggested that 2 is a highly unique endogenous compound that induces ROS accumulation specifically in Samanea extensor motor cells. Furthermore, this 2-induced ROS accumulation was effectively inhibited by diphenyleneiodonium chloride, an inhibitor of NADPH oxidases, although it may also affect other enzymatic systems.⁹⁵⁾⁾ Although this suggests that ROS production likely involves respiratory burst oxidase homologues (Rboh) proteins, which are plant NADPH oxidases, alternative mechanisms of ROS generation induced by 2 cannot be completely excluded. Based on these findings, we proposed a mechanistic model in which 2 binds to the unidentified membrane protein⁷⁴⁾⁾ and subsequently activates Rboh through a signaling cascade that remains to be fully elucidated, ultimately leading to ROS accumulation. Notably, the ROS-mediated cell shrinkage was observed exclusively during daylight hours when SPORK2 protein expression was detectable. Corroborating these observations, heterologously expressed SPORK2 in Xenopus oocytes exhibited significantly enhanced K⁺ transport activity upon exogenous application of H₂O₂, a predominant ROS species (Fig. 9B). In contrast, JAG did not affect the ion transport activity of SsSLAH1-SsSLAH3 complex. These findings collectively suggest a mechanism whereby 2 induces cellular shrinkage by stimulating SPORK2-mediated K⁺ efflux through ROS-dependent activation (Fig. 9A). This redox sensitivity parallels that observed in Arabidopsis SKOR, which is similarly activated by H₂O₂ through a mechanism requiring a specific cysteine residue.⁹⁶⁾⁾ The cysteine residue is also conserved in SPORK2 (Cys182), suggesting a similar ROS-dependent regulation. This result demonstrated that 2 induce leaf-folding of S. saman through ROS-mediated activation of SPORK2 channel.

Figure 9.

Possible molecular mechanism of LCF-induced leaf folding in Samanea saman. (A) Schematic representation of the proposed mechanism for leaf folding induced by LCF (2). LCF targets extensor cells to cause ROS accumulation. This results in the activation of K⁺ efflux through SPORK2. Cl⁻ efflux by SsSLAH3 and/or other anion channels may be achieved through a still-unknown mechanism. (B) Electrophysiological results of SPORK2 activation by ROS (10 mM H₂O₂). SPORK2 was heterologously expressed in Xenopus laevis oocytes and K⁺ current was recorded by the two-electrode voltage-clamp method.

9. “Rain tree” phenomenon: SPORK2 as a temperature sensor of a plant

Samanea exhibits not only nyctinastic leaf movements but also responds to rainfall by folding its leaves (Fig. 10A).⁹⁷⁾⁾ This is why it is commonly called the “rain tree”. Although the physiological basis of this intriguing phenomenon had remained elusive, Ueda et al. revealed that changes in leaf surface temperature induced by raindrops serve as the primary trigger for this response.⁹⁸⁾⁾ Ueda et al. also demonstrated that low-temperature stimuli elicit cellular swelling and deactivation of K⁺_out current in flexor motor cells (Fig. 10B and 10C). Consistent with these observations, SPORK2 heterologously expressed in Xenopus oocytes displayed pronounced temperature sensitivity, exhibiting enhanced activity at elevated temperatures (30 ℃) and diminished function at lower temperatures (18 ℃) (Fig. 10D). Notably, the Cl⁻ transport activity of the SsSLAH1-SsSLAH3 complex, which co-localizes with SPORK2 in flexor cells, showed no discernible temperature dependence in Xenopus oocytes (Fig. 10E). These findings suggested a mechanism whereby rain-induced cooling inactivates SPORK2, attenuating K⁺ efflux and consequently promoting flexor motor cell swelling that drives leaf folding (Fig. 10B).

Figure 10.

Possible molecular mechanism of “Rain Tree” phenomenon in Samanea saman. (A) Photographs of Samanea before and after folding its leaves due to rain (“Rain Tree” phenomenon). (B) Schematic representation of the proposed mechanism for rain-induced leaf folding. Rain decreases leaf temperature and this deactivates SPORK2 (i). The resulting K⁺ accumulation will inhibit SsSLAH1-SsSLAH3 to maintain the ion balance inside the cells (ii). Due to the KCl accumulation, flexor cells swell. (C) Temperature-dependent K⁺ current recorded in flexor motor cells using the patch-clamp technique. (D and E) Ion transport activities of SPORK2 (D) and SsSLAH1-SsSLAH3 (E) heterologously expressed in Xenopus laevis oocytes. SPORK2 shows temperature-dependent K⁺ currents, but SsSLAH1-SsSLAH3 shows temperature-independent Cl⁻ transport activity. Ion currents were recorded by the two-electrode voltage-clamp method.

To establish the genetic basis for SPORK2-mediated temperature-dependent regulation of cell volume, they employed Arabidopsis guard cells as a genetically tractable model system, circumventing the transformation limitations of Samanea motor cells. Arabidopsis guard cells express GORK, a SPORK2 ortholog that facilitates stomatal closure by mediating K⁺ efflux and subsequent cell shrinkage.^99),100)) When we introduced SPORK2 into Arabidopsis gork mutants, the transgenic plants maintained the capacity for dark-induced stomatal closure in high-temperature conditions (22 ℃) and also impaired closure in response to low-temperature treatment (5 ℃). These genetic complementation studies provided compelling in planta evidence that SPORK2, unlike GORK, undergoes temperature-dependent inactivation at lower temperatures, thereby failing to induce cellular shrinkage.

Notably, GORK expressed in Xenopus oocytes exhibited temperature sensitivity inverse to that of SPORK2, showing slight activation at 18 ℃ relative to 30 ℃.⁹⁸⁾⁾ Remarkably, SPORK2 orthologs from two other leguminous species, including G. max (GmORK1) and Medicago truncatula (MtGORK), as well as SKOR, another K⁺_out channel in Arabidopsis, all exhibited similar temperature-dependent activation profiles with enhanced activity at elevated temperatures.⁹⁸⁾⁾ This phylogenetic conservation suggested that SPORK2 orthologs may function as evolutionarily conserved temperature sensors across various plant taxa, potentially playing fundamental roles in adaptive responses to environmental temperature fluctuations.

These findings revealed a fascinating dual regulatory system for leaf movement in S. saman, where both endogenous chemical factors and exogenous environmental stimuli converge on the same molecular effector (SPORK2) but through distinct cellular targets and mechanisms. The LCF (2) pathway represents an endogenous mechanism under biological clock control, acting specifically on extensor cells through ROS-mediated SPORK2 activation. This likely serves as a background mechanism that consistently regulates daily leaf-movement patterns. In contrast, the temperature-sensing pathway represents an exogenous mechanism that responds to environmental changes such as rainfall, acting on flexor cells through temperature-dependent inactivation of SPORK2. Although both pathways ultimately result in leaf folding, they operate through completely different cellular targets and molecular mechanisms. Intriguingly, morning leaf opening involves yet another mechanism, where the SsSLAH1-SsSLAH3 complex forms exclusively in flexor cells, activating SPORK2 through membrane depolarization. This complex interplay of multiple regulatory pathways with SPORK2 as a central integrator suggests a sophisticated evolutionary adaptation that allows Samanea to respond appropriately to both predictable daily cycles and unpredictable environmental changes. However, several aspects of these mechanisms remain to be elucidated, including how LCF affects Cl⁻ channels (presumably SsSLAH3) and how low temperature influences the SsSLAH1-SsSLAH3 complex. Figure 11 summarizes these parallel pathways, highlighting both the established mechanisms and the aspects that require further investigation.

Figure 11.

Summary of leaf-folding mechanisms based on the differences in target cells between LCF and rain. In the morning, the SsSLAH1-SsSLAH3 complex forms exclusively in flexor cells, activating SPORK2 through depolarization and causing the leaves to open. LCF (2) acts specifically on extensor cells, activating SPORK2 through ROS to induce leaf folding. In contrast, rain (low temperature) inactivates SPORK2 in flexor cells, resulting in leaf folding. However, please note that the mechanisms by which LCF activates chloride channels and low temperature inactivates the SsSLAH1+SsSLAH3 complex remain unknown.

Transient receptor potential (TRP) channels and their homologs are temperature-sensitive ion channels widely distributed in organisms, whereas plant do not have TRP-like genes and their temperature-sensing mechanisms have long been unknown.¹⁰¹⁾⁾ Calcium channels, photoreceptors, and circadian clock components have been proposed as potential contributors to temperature-sensing mechanisms in plants Among them, cyclic nucleotide-gated channels (CNGCs), particularly CNGC2 and CNGC6, have attracted attention as candidates that mediate temperature-dependent calcium influx and initiate calcium-dependent signaling cascades.^102),103)) In parallel, phytochromes, red/far-red light photoreceptors traditionally associated with light signaling, have been implicated as potential thermosensors based on their temperature-sensitive biochemical properties.^104),105)) EARLY FLOWERING 3 (ELF3), a core component of the evening complex within the circadian clock, has also been suggested to function in temperature perception.¹⁰⁶⁾⁾ Despite these recent advances, definitive experimental evidence supporting their roles as primary temperature sensors remains limited.¹⁰¹⁾⁾ This underscores the urgent need for innovative experimental strategies capable of distinguishing primary thermosensory events from downstream signaling processes. The structures of animal TRP channels are well-established by cryo-EM.¹⁰⁷⁾⁾ Despite these structural advances, the precise molecular mechanism of temperature sensing remains controversial.¹⁰⁸⁾⁾ Comparative analysis of temperature sensing mechanisms between plant SPORK2 and animal TRP channels could provide fascinating evolutionary insights into how these phylogenetically distant organisms have independently evolved molecular temperature sensors with similar functional outputs but potentially distinct structural solutions.

The long journey on nyctinasty from identification of endogenous chemical factors and subsequent elucidation of molecular players delivered an unanticipated gift, the discovery of a molecular mechanism of temperature-sensing in plant.

Profile

Minoru Ueda was born in Nagoya, Japan, in 1965. He received his B.Sc. degree from the Department of Chemistry, Faculty of Science, Konan University in 1989 and earned his Ph.D. from Nagoya University in 1994 under the supervision of Professors Toshio Goto and Minoru Isobe, focusing on the supramolecular blue pigment of cornflower (Centaurea cyanus). He subsequently joined the Department of Chemistry, Keio University. In 2004, he was appointed full professor at Tohoku University. From 2011 to 2016, he served as the representative of the MEXT Grant-in-Aid for Scientific Research on Innovative Areas, “Chemical Biology of Natural Products”, and subsequently as the representative of the JSPS A3 Foresight Program. He is now an Advisor of the Japanese Society for Chemical Biology, director of Suntory Foundation for Life Science, and Advisory Board Committee Member of the IUPAC International Symposium on the Chemistry of Natural Products and the International Conference on Biodiversity (ISCNP& ICOB). He is the organizer of IUPAC 33rd ISCNP& 13th ICOB in 2027. His research interests lie in the bioorganic chemistry of plant movement and the chemical biology of oxylipin plant hormones. He has received several awards, including the Award for Young Chemists from the Chemical Society of Japan (2001), the Yomiuri Techno-Forum Gold Medal Prize from the Yomiuri Shimbun (2003), and the JSCRP Award from the Japanese Society for Chemical Regulation of Plants (2014).

Conflict of interest

The authors declare no conflicts of interest.