Mechanism of the Pulvinus-Driven Leaf Movement: An Overview.

Leaf movement is a manifestation of plant response to the changing internal and external environment, aiming to optimize plant growth and development. Leaf movement is usually driven by a specialized motor organ, the pulvinus, and this movement is associated with different changes in volume and expansion on the two sides of the pulvinus. Blue light, auxin, GA, H+-ATPase, K+, Cl-, Ca2+, actin, and aquaporin collectively influence the changes in water flux in the tissue of the extensor and flexor of the pulvinus to establish a turgor pressure difference, thereby controlling leaf movement. However, how these factors regulate the multicellular motility of the pulvinus tissues in a species remains obscure. In addition, model plants such as Medicago truncatula, Mimosa pudica, and Samanea saman have been used to study pulvinus-driven leaf movement, showing a similarity in their pulvinus movement mechanisms. In this review, we summarize past research findings from the three model plants, and using Medicago truncatula as an example, suggest that genes regulating pulvinus movement are also involved in regulating plant growth and development. We also propose a model in which the variation of ion flux and water flux are critical steps to pulvinus movement and highlight questions for future research.

Simultaneous analysis of shape and internal structure of a curved Hibiscus cannabinus pulvinus: X-ray microtomography and semi-automated quantification.

In the Malvaceae family, dynamic solar tracking by leaves is actuated by the deformation of the pulvinus, a thickened region at the leaf blade-petiole junction. While the internal structure is believed to play a crucial role in this process, experimental verification has been challenging due to technical limitations. To address this gap, we developed a semi-automated workflow, which integrates data analysis and image processing to simultaneously analyze the shape and internal structure of a Malvaceae pulvinus using X-ray microtomography. Firstly, we found that kenaf (Hibiscus cannabinus L.), a Malvaceae species with curved pulvini, exhibited solar-tracking leaf movement and selected it as a model system. We employed diffusible iodine-based contrast-enhanced computed tomography to visualize the internal structure of the kenaf pulvinus. Analysis of the pulvini's shape revealed variations in pulvinus morphology, yet plausible prediction of the centerline was accomplished using polar polynomial regression. Upon slicing the pulvini perpendicular to the centerline, we observed distinct gray value gradients along the proximo-distal and adaxial-abaxial axes, challenging threshold-based tissue segmentation. This workflow successfully generated three modified 3D images and derived quantitative parameters. Using these quantitative parameters, we conducted network analysis and found the linkage between the size-normalized cortex cross-sectional area and curvature. Polynomial least absolute shrinkage and selection operator (LASSO) regression revealed the relationship between the size-normalized cortex cross-sectional area and curvature commonly in all three tested samples. This workflow enables simultaneous analysis of the shape and internal structure, significantly improving the reproducibility of Malvaceae leaf pulvinus characterization.

Nyctinastic movement in legumes: Developmental mechanisms, factors and biological significance.

In legumes, a common phenomenon known as nyctinastic movement is observed. This movement involves the horizontal expansion of leaves during the day and relative vertical closure at night. Nyctinastic movement is driven by the pulvinus, which consists of flexor and extensor motor cells. The turgor pressure difference between these two cell types generates a driving force for the bending and deformation of the pulvinus. This review focuses on the developmental mechanisms of the pulvinus, the factors affecting nyctinastic movement, and the biological significance of this phenomenon in legumes, thus providing a reference for further research on nyctinastic movement.

Dysfunction of an Anaphase-Promoting Complex Subunit 8 Homolog Leads to Super-Short Petioles and Enlarged Petiole Angles in Soybean.

Plant height, petiole length, and the angle of the leaf petiole and branch angles are crucial traits determining plant architecture and yield in soybean (Glycine max L.). Here, we characterized a soybean mutant with super-short petioles (SSP) and enlarged petiole angles (named Gmssp) through phenotypic observation, anatomical structure analysis, and bulk sequencing analysis. To identify the gene responsible for the Gmssp mutant phenotype, we established a pipeline involving bulk sequencing, variant calling, functional annotation by SnpEFF (v4.0e) software, and Integrative Genomics Viewer analysis, and we initially identified Glyma.11G026400, encoding a homolog of Anaphase-promoting complex subunit 8 (APC8). Another mutant, t7, with a large deletion of many genes including Glyma.11G026400, has super-short petioles and an enlarged petiole angle, similar to the Gmssp phenotype. Characterization of the t7 mutant together with quantitative trait locus mapping and allelic variation analysis confirmed Glyma.11G026400 as the gene involved in the Gmssp phenotype. In Gmssp, a 4 bp deletion in Glyma.11G026400 leads to a 380 aa truncated protein due to a premature stop codon. The dysfunction or absence of Glyma.11G026400 caused severe defects in morphology, anatomical structure, and physiological traits. Transcriptome analysis and weighted gene co-expression network analysis revealed multiple pathways likely involved in these phenotypes, including ubiquitin-mediated proteolysis and gibberellin-mediated pathways. Our results demonstrate that dysfunction of Glyma.11G026400 leads to diverse functional consequences in different tissues, indicating that this APC8 homolog plays key roles in cell differentiation and elongation in a tissue-specific manner. Deciphering the molecular control of petiole length and angle enriches our knowledge of the molecular network regulating plant architecture in soybean and should facilitate the breeding of high-yielding soybean cultivars with compact plant architecture.

A touchy subject: Ca2+ signaling during leaf movements in Mimosa.

Mimosa pudica, the sensitive plant, responds to stimuli such as touch and wounding with leaf movements that propagate throughout the plant. The motion is driven by changes in the turgor of specialized cells in a set of motor organs called pulvinae. By imaging cellular Ca2+ levels as the wave of movement propagates through the leaf, Hagihara and colleagues now show that Ca2+ signals precede and predict the pulvinar movements. These results provide compelling support for a model where Mimosa uses a Ca2+-related response system to trigger its leaf movements. These researchers then used CRISPR to delete a critical genetic regulator of pulvinar development, producing plants with immobile leaves. These plants experienced more herbivory than wild type, suggesting that the Ca2+-triggered leaf movements are an adaptation to deter herbivory.

Multiscale structural anisotropy steers plant organ actuation.

Leaf movement in vascular plants is executed by joint-like structures called pulvini. Many structural features of pulvini have been described at subcellular, cellular, and tissue scales of organization; however, how the characteristic hierarchical architecture of plant tissue influences pulvinus-mediated actuation remains poorly understood. To investigate the influence of multiscale structure on turgor-driven pulvinus movements, we visualized Mimosa pudica pulvinus morphology and anatomy at multiple hierarchical scales of organization and used osmotic perturbations to experimentally swell pulvini in incremental states of dissection. We observed directional cellulose microfibril reinforcement, oblong, spindle-shaped primary pit fields, and longitudinally slightly compressed cell geometries in the parenchyma of M. pudica. Consistent with these observations, isolated parenchyma tissues displayed highly anisotropic swelling behaviors indicating a high degree of mechanical anisotropy. Swelling behaviors at higher scales of pulvinus organization were also influenced by the presence of the pulvinus epidermis, which displayed oblong epidermal cells oriented transverse to the pulvinus long axis. Our findings indicate that structural specializations spanning multiple hierarchical scales of organization guide hydraulic deformation of pulvini, suggesting that multiscale mechanics are crucial to the translation of cell-level turgor variations into organ-scale pulvinus motion in vivo.

Anatomical study on the developmental process of the swollen internodes of Phryma (Phrymaceae, eudicots).

MAIN CONCLUSION: We discovered that the internodal swellings of Phryma (eudicots) stems were same as the internodal pulvini of Poaceae (monocots) from the viewpoints of internal structures and functions. The stems of eudicots are usually rod-shaped and are composed of nodes, attached by leaves, and internodes. The internodes of some species, belonging to the clade 'asterids' and its sister clade 'Caryophyllales' of eudicots, have swellings, which have negative tropism, at the basal or apical part of each internode. To know the internal features of the swollen internodes, we performed outer morphological and anatomical studies on the swollen internodes of Phryma, eudicots, one of the genera having swollen internodes, from the winter bud stage to the flowering stage. The results revealed the following: (i) the swollen regions of the internodes were composed of less lignified tissues (e.g., endodermis without Casparian strips, and xylem having less lignified xylem fibers); (ii) the internodal less lignified parts were supported by collenchyma; (iii) the endodermis includes amyloplasts, having accumulated starch granules, which would function as statoliths for negative gravitropism. Consequently, we determined that the swollen parts of the Phryma internodes are same as the internodal pulvini of Poaceae of monocots from the viewpoints of internal structures and functions.

Mechanics of Reversible Deformation during Leaf Movement and Regulation of Pulvinus Development in Legumes.

Plant cell deformation is a mechanical process that is driven by differences in the osmotic pressure inside and outside of the cell and is influenced by cell wall properties. Legume leaf movements result from reversible deformation of pulvinar motor cells. Reversible cell deformation is an elastic process distinct from the irreversible cell growth of developing organs. Here, we begin with a review of the basic mathematics of cell volume changes, cell wall function, and the mechanics of bending deformation at a macro scale. Next, we summarize the findings of recent molecular genetic studies of pulvinar development. We then review the mechanisms of the adaxial/abaxial patterning because pulvinar bending deformation depends on the differences in mechanical properties and physiological responses of motor cells on the adaxial versus abaxial sides of the pulvinus. Intriguingly, pulvini simultaneously encompass morphological symmetry and functional asymmetry along the adaxial/abaxial axis. This review provides an introduction to leaf movement and reversible deformation from the perspective of mechanics and molecular genetics.

Deformation and mechanics of a pulvinus-inspired material.

Mimosa pudicarapidly folds leaves when touched. Motion is created by pulvini, 'the plant muscles' that allow plants to produce various complex motions. Plants rely on local control of the turgor pressure to create on-demand motion. In this paper, the mechanics of a cellular material inspired from pulvinus ofM. pudicais studied. First, the manufacturing process of a cell-controllable material is described. Its deformation behaviour when pressured is tested, focusing on three pressure patterns of reference. The deformations are modelled based on the minimisation of elastic energy framework. Depending on pressurisation pattern and magnitude, reversible buckling-induced motion may occur.

Multidimensional Gene Regulatory Landscape of Motor Organ Pulvinus in the Model Legume Medicago truncatula.

Nyctinastic leaf movement of Fabaceae is driven by the tiny motor organ pulvinus located at the base of the leaf or leaflet. Despite the increased understanding of the essential role of ELONGATED PETIOLULE1 (ELP1)/PETIOLE LIKE PULVINUS (PLP) orthologs in determining pulvinus identity in legumes, key regulatory components and molecular mechanisms underlying this movement remain largely unclear. Here, we used WT pulvinus and the equivalent tissue in the elp1 mutant to carry out transcriptome and proteome experiments. The omics data indicated that there are multiple cell biological processes altered at the gene expression and protein abundance level during the pulvinus development. In addition, comparative analysis of different leaf tissues provided clues to illuminate the possible common primordium between pulvinus and petiole, as well as the function of ELP1. Furthermore, the auxin pathway, cell wall composition and chloroplast distribution were altered in elp1 mutants, verifying their important roles in pulvinus development. This study provides a comprehensive insight into the motor organ of the model legume Medicago truncatula and further supplies a rich dataset to facilitate the identification of novel players involved in nyctinastic movement.

The F-box protein MIO1/SLB1 regulates organ size and leaf movement in Medicago truncatula.

The size of leaf and seed organs, determined by the interplay of cell proliferation and expansion, is closely related to the final yield and quality of forage and crops. Yet the cellular and molecular mechanisms underlying organ size modulation remain poorly understood, especially in legumes. Here, MINI ORGAN1 (MIO1), which encodes an F-box protein SMALL LEAF AND BUSHY1 (SLB1) recently reported to control lateral branching in Medicago truncatula, was identified as a key regulator of organ size. We show that loss-of-function of MIO1/SLB1 severely reduced organ size. Conversely, plants overexpressing MIO1/SLB1 had enlarged organs. Cellular analysis revealed that MIO1/SLB1 controlled organ size mainly by modulating primary cell proliferation during the early stages of leaf development. Biochemical analysis revealed that MIO1/SLB1 could form part of SKP1/Cullin/F-box (SCF) E3 ubiquitin ligase complex, to target BIG SEEDS1 (BS1), a repressor of primary cell division, for degradation. Interestingly, we found that MIO1/SLB1 also played a key role in pulvinus development and leaf movement by modulating cell proliferation of the pulvinus as leaves developed. Our study not only demonstrates a conserved role of MIO1/SLB1 in the control of organ size in legumes, but also sheds light on the novel function of MIO1/SLB1 in leaf movement.

The geometry of the compound leaf plays a significant role in the leaf movement of Medicago truncatula modulated by mtdwarf4a.

In most legumes, two typical features found in leaves are diverse compound forms and the pulvinus-driven nyctinastic movement. Many genes have been identified for leaf-shape determination, but the underlying nature of leaf movement as well as its association with the compound form remains largely unknown. Using forward-genetic screening and whole-genome resequencing, we found that two allelic mutants of Medicago truncatula with unclosed leaflets at night were impaired in MtDWARF4A (MtDWF4A), a gene encoding a cytochrome P450 protein orthologous to Arabidopsis DWARF4. The mtdwf4a mutant also had a mild brassinosteroid (BR)-deficient phenotype bearing pulvini without significant deficiency in organ identity. Both mtdwf4a and dwf4 could be fully rescued by MtDWF4A, and mtdwf4a could close their leaflets at night after the application of exogenous 24-epi-BL. Surgical experiments and genetic analysis of double mutants revealed that the failure to exhibit leaf movement in mtdwf4a is a consequence of the physical obstruction of the overlapping leaflet laminae, suggesting a proper geometry of leaflets is important for their movement in M. truncatula. These observations provide a novel insight into the nyctinastic movement of compound leaves, shedding light on the importance of open space for organ movements in plants.

Functional characterization of PETIOLULE-LIKE PULVINUS (PLP) gene in abscission zone development in Medicago truncatula and its application to genetic improvement of alfalfa.

Alfalfa (Medicago sativa L.) is one of the most important forage crops throughout the world. Maximizing leaf retention during the haymaking process is critical for achieving superior hay quality and maintaining biomass yield. Leaf abscission process affects leaf retention. Previous studies have largely focused on the molecular mechanisms of floral organ, pedicel and seed abscission but scarcely touched on leaf and petiole abscission. This study focuses on leaf and petiole abscission in the model legume Medicago truncatula and its closely related commercial species alfalfa. By analysing the petiolule-like pulvinus (plp) mutant in M. truncatula at phenotypic level (breakstrength and shaking assays), microscopic level (scanning electron microscopy and cross-sectional analyses) and molecular level (expression level and expression pattern analyses), we discovered that the loss of function of PLP leads to an absence of abscission zone (AZ) formation and PLP plays an important role in leaflet and petiole AZ differentiation. Microarray analysis indicated that PLP affects abscission process through modulating genes involved in hormonal homeostasis, cell wall remodelling and degradation. Detailed analyses led us to propose a functional model of PLP in regulating leaflet and petiole abscission. Furthermore, we cloned the PLP gene (MsPLP) from alfalfa and produced RNAi transgenic alfalfa plants to down-regulate the endogenous MsPLP. Down-regulation of MsPLP results in altered pulvinus structure with increased leaflet breakstrength, thus offering a new approach to decrease leaf loss during alfalfa haymaking process.

The bHLH transcription factor PPLS1 regulates the color of pulvinus and leaf sheath in foxtail millet (Setaria italica).

KEY MESSAGE: The bHLH transcription factor, PPLS1, interacts with SiMYB85 to control the color of pulvinus and leaf sheath by regulating anthocyanin biosynthesis in foxtail millet (Setaria italica). Foxtail millet (Setaria italica), a self-pollinated crop with numerous small florets, is difficult for cross-pollination. The color of pulvinus and leaf sheath with purple being dominant to green is an indicative character and often used for screening authentic hybrids in foxtail millet crossing. Deciphering molecular mechanism controlling this trait would greatly facilitate genetic improvement of cultivars in foxtail millet. Here, using the F2 bulk specific-locus amplified fragment sequencing approach, we mapped the putative causal gene for the purple color of pulvinus and leaf sheath (PPLS) trait to a 100 Kb region on chromosome 7. Expression analyses of the 15 genes in this region revealed that Seita.7G195400 (renamed here as PPLS1) was differentially expressed between purple and green cultivars. PPLS1 encodes a bHLH transcription factor and is localized in the nucleus with a transactivation activity. Furthermore, we observed that expression of a MYB transcription factor gene, SiMYB85 (Seita.4G086300) involved in anthocyanin biosynthesis, shows a totally positive association with that of PPLS1. Heterologous co-expression of both PPLS1 and SiMYB85 in tobacco leaves led to elevated anthocyanin accumulation and expression of some anthocyanin-related genes. Furthermore, PPLS1 physically interacts with SiMYB85. Taken together, our results suggest that PPLS1 interacts with SiMYB85 to control the color of pulvinus and leaf sheath by regulating anthocyanin biosynthesis in foxtail millet.

Characteristic early membrane effects induced by tryptophan in pulvinar motor cells.

Tryptophan at concentrations higher than 0.1 mM, triggered characteristic early physiological effects such as rapid (within 5 min) dose-dependent membrane hyperpolarization in Mimosa pudica motor cells and modification of the time course of the spontaneous proton efflux monitored in the incubation medium of pulvinar tissues. The rapid modifications of the leaf turgor-mediated movements seen on the primary pulvini of M. pudica following a shock and on Cassia fasciculata leaflets during a transition from light to darkness indicate that tryptophan disturbed the ionic migrations involved in the electrophysiological events and in the osmocontractile reaction of the motor cells. These reactions were specific to tryptophan compared to those induced by serine and 5-hydroxytryptophan. The tryptophan mode of action cannot be linked to a direct modification of the plasma membrane H+-ATPase activity as monitored on purified pulvinar plasma membrane vesicles. The tryptophan metabolism-linked products tryptamine and indole also inhibited the motile reactions, activated in a continuous manner the H+ secretion of pulvinar tissues and showed properties of a protonophore and an ATPase activity inhibitor on plasma membrane vesicles, respectively. The specific behavior of tryptophan in the reaction studies here is discussed in light of the previously reported action of phytohormones.

Characterization of the grapevine Shaker K+ channel VvK3.1 supports its function in massive potassium fluxes necessary for berry potassium loading and pulvinus-actuated leaf movements.

In grapevine, climate changes lead to increased berry potassium (K+ ) contents that result in must with low acidity. Consequently, wines are becoming 'flat' to the taste, with poor organoleptic properties and low potential aging, resulting in significant economic loss. Precise investigation into the molecular determinants controlling berry K+ accumulation during its development are only now emerging. Here, we report functional characterization by electrophysiology of a new grapevine Shaker-type K+ channel, VvK3.1. The analysis of VvK3.1 expression patterns was performed by qPCR and in situ hybridization. We found that VvK3.1 belongs to the AKT2 channel phylogenetic branch and is a weakly rectifying channel, mediating both inward and outward K+ currents. We showed that VvK3.1 is highly expressed in the phloem and in a unique structure located at the two ends of the petiole, identified as a pulvinus. From the onset of fruit ripening, all data support the role of the VvK3.1 channel in the massive K+ fluxes from the phloem cell cytosol to the berry apoplast during berry K+ loading. Moreover, the high amount of VvK3.1 transcripts detected in the pulvinus strongly suggests a role for this Shaker in the swelling and shrinking of motor cells involved in paraheliotropic leaf movements.

RNA silencing in the life cycle of soybean: multiple restriction systems and spatiotemporal variation associated with plant architecture.

The expression of transgenes introduced into a plant genome is sometimes suppressed by RNA silencing. Although local and systemic spread of RNA silencing have been studied, little is known about the mechanisms underlying spatial and temporal variation in transgene silencing between individual plants or between plants of different generations, which occurs seemingly stochastically. Here, we analyzed the occurrence, spread, and transmission of RNA silencing of the green fluorescent protein (GFP) gene over multiple generations of the progeny of a single soybean transformant. Observation of GFP fluorescence in entire plants of the T3-T5 generations indicated that the initiation and subsequent spread of GFP silencing varied between individuals, although this GFP silencing most frequently began in the primary leaves. In addition, GFP silencing could spread into the outer layer of seed coat tissues but was hardly detectable in the embryos. These results are consistent with the notion that transgene silencing involves its reset during reproductive phase, initiation after germination, and systemic spread in each generation. GFP silencing was absent in the pulvinus, suggesting that its cortical cells inhibit cell-to-cell spread or induction of RNA silencing. The extent of GFP silencing could differ between the stem and a petiole or between petiolules, which have limited vascular bundles connecting them and thus deter long-distant movement of silencing. Taken together, these observations indicate that the initiation and/or spread of RNA silencing depend on specific features of the architecture of the plant in addition to the mechanisms that can be conserved in higher plants.

Leaf movements and their relationship with the lunisolar gravitational force.

BACKGROUND: Observation of the diurnal ascent and descent of leaves of beans and other species, as well as experimental interventions into these movements, such as exposures to light at different times during the movement cycle, led to the concept of an endogenous 'clock' as a regulator of these oscillations. The physiological basis of leaf movement can be traced to processes that modulate cell volume in target tissues of the pulvinus and petiole. However, these elements of the leaf-movement process do not completely account for the rhythms that are generated following germination in constant light or dark conditions, or when plants are transferred to similar free-running conditions. SCOPE: To develop a new perspective on the regulation of leaf-movement rhythms, many of the published time courses of leaf movements that provided evidence for the concept of the endogenous clock were analysed in conjunction with the contemporaneous time courses of the lunisolar tidal acceleration at the relevant experimental locations. This was made possible by application of the Etide program, which estimates, with high temporal resolution, local gravitational changes as a consequence of the diurnal variations of the lunisolar gravitational force due to the orbits and relative positions of Earth, Moon and Sun. In all cases, it was evident that a synchronism exists between the times of the turning points of both the lunisolar tide and of the leaftide when the direction of leaf movement changes. This finding of synchrony leads to the hypothesis that the lunisolar tide is a regulator of the leaftide, and that the rhythm of leaf movement is not necessarily of endogenous origin but is an expression of an exogenous lunisolar 'clock' impressed upon the leaf-movement apparatus. CONCLUSIONS: Correlation between leaftide and Etide time courses holds for leaf movement rhythms in natural conditions of the greenhouse, in conditions of constant light or dark, under microgravity conditions of the International Space Station, and also holds for rhythms that are atypical, such as pendulum and relaxation rhythms whose periods are longer or shorter than usual. Even the apparently spontaneous short-period, small-amplitude rhythms recorded from leaves under unusual growth conditions are consistent with the hypothesis of a lunisolar zeitgeber. Two hypotheses that could account for the synchronism between leaftide and Etide, and which are based on either quantum considerations or on classical Newtonian physics, are presented and discussed.

PHANTASTICA regulates leaf polarity and petiole identity in Medicago truncatula.

Establishment of proper polarities along the adaxial-abaxial, proximodistal, and medial-lateral axes is a critical step for the expansion of leaves from leaf primordia. It has been shown that the MYB domain protein, asymmetric leaves1/rough sheath2/PHANTASTICA (collectively named ARP) plays an important role in this process. Loss of function of ARP leads to severe leaf polarity defects, such as abaxialized or needle-like leaves. In addition to its role in leaf polarity establishment, we have recently shown that the Medicago truncatula ARP gene, MtPHAN, also plays a role in leaf petiole identity regulation. We show that a mutation of MtPHAN results in petioles acquiring characteristics of the motor organ, pulvinus, including small epidermal cells with extensive cell surface modifications and altered vascular tissue development. Taken together, our results reveal a previously unidentified function of ARP in leaf development.

Uptake and metabolic effects of salicylic acid on the pulvinar motor cells of Mimosa pudica L.

In this paper, the salicylic acid (o-hydroxy benzoic acid) (SA) uptake by the pulvinar tissues of Mimosa pudica L. pulvini was shown to be strongly pH-dependent, increasing with acidity of the assay medium. This uptake was performed according to a unique affinity system (K(m) = 5.9 mM, V(m) = 526 pmol mgDW(-1)) in the concentration range of 0.1-5 mM. The uptake rate increased with increasing temperature (5-35 °C) and was inhibited following treatment with sodium azide (NaN3) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), suggesting the involvement of an active component. Treatment with p-chloromercuribenzenesulfonic acid (PCMBS) did not modify the uptake, indicating that external thiol groups were not necessary. KCl, which induced membrane depolarization had no significant effect, and fusicoccin (FC), which hyperpolarized cell membrane, stimulated the uptake, suggesting that the pH component of the proton motive force was likely a driving force. These data suggest that the SA uptake by the pulvinar tissues may be driven by two components: an ion-trap mechanism playing a pivotal role and a putative carrier-mediated mechanism. Unlike other benzoic acid derivatives acting as classical respiration inhibitors (NaN3 and KCN), SA modified the pulvinar cell metabolism by increasing the respiration rate similar to CCCP and 2,4-dinitrophenol (DNP). Furthermore, SA inhibited the osmoregulated seismonastic reaction in a pH dependent manner and induced characteristic damage to the ultrastructural features of the pulvinar motor cells, particularly at the mitochondrial level.