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.

Calcium changes in Robinia pseudoacacia pulvinar motor cells during nyctinastic closure mediated by phytochromes.

Potassium pyroantimonate precipitation, transmission electron microscopy, and X-ray microanalysis were used to investigate the subcellular localization of loosely bound calcium in Robinia pseudoacacia pulvinar motor cells during phytochrome-mediated nyctinastic closure. Calcium localization was carried out in pulvini collected in white light 2 h after the beginning of the photoperiod, immediately after a red light or a far-red light pulse applied 2 h after the beginning of the photoperiod and after 15 or 25 min of darkness respectively. Calcium antimonate precipitates were found in all the pulvinar tissues from the epidermis to the vascular bundle, independent of the light treatment. At subcellular level, precipitates were found mainly in the intercellular spaces, the inner surface of the plasma membrane, cytoplasm, colloidal vacuoles, and nuclei. Red light enhanced the nyctinastic closure of leaflets and caused an asymmetric distribution of cytosolic calcium precipitates between the extensor and flexor motor cells. Both the number and area of the cytosolic calcium precipitates drastically increased in the extensor cells compared to the flexor motor cells. Red light had a rapid and transient effect on the distribution of cytosolic calcium precipitates, which occurred during or at the end of the irradiation, before leaflet closure. By contrast, the distribution of cytosolic loosely bound calcium was similar between the extensor and flexor motor cells after irradiation with far-red light. Our results demonstrate that red light causes specific calcium mobilization in pulvinar motor cells and suggest the involvement of cytoplasmic Ca2+ as a second messenger for phytochrome during nyctinastic closure.

Real-time imaging of pulvinus bending in Mimosa pudica.

Mimosa pudica is a plant that rapidly shrinks its body in response to external stimuli. M. pudica does not perform merely simple movements, but exhibits a variety of movements that quickly change depending on the type of stimuli. Previous studies have investigated the motile mechanism of the plants from a biochemical perspective. However, an interdisciplinary study on the structural characteristics of M. pudica should be accompanied by biophysical research to explain the principles underlying such movements. In this study, the structural characteristics and seismonastic reactions of M. pudica were experimentally investigated using advanced bio-imaging techniques. The results show that the key factors for the flexible movements by the pulvinus are the following: bendable xylem bundle, expandable/shrinkable epidermis, tiny wrinkles for surface modification, and a xylem vessel network for efficient water transport. This study provides new insight for better understanding the M. pudica motile mechanism through structural modification.

Conserved genetic determinant of motor organ identity in Medicago truncatula and related legumes.

Plants exhibit various kinds of movements that have fascinated scientists and the public for centuries. Physiological studies in plants with the so-called motor organ or pulvinus suggest that cells at opposite sides of the pulvinus mediate leaf or leaflet movements by swelling and shrinking. How motor organ identity is determined is unknown. Using a genetic approach, we isolated a mutant designated elongated petiolule1 (elp1) from Medicago truncatula that fails to fold its leaflets in the dark due to loss of motor organs. Map-based cloning indicated that ELP1 encodes a putative plant-specific LOB domain transcription factor. RNA in situ analysis revealed that ELP1 is expressed in primordial cells that give rise to the motor organ. Ectopic expression of ELP1 resulted in dwarf plants with petioles and rachises reduced in length, and the epidermal cells gained characteristics of motor organ epidermal cells. By identifying ELP1 orthologs from other legume species, namely pea (Pisum sativum) and Lotus japonicus, we show that this motor organ identity is regulated by a conserved molecular mechanism.

Pulvinus functional traits in relation to leaf movements: a light and transmission electron microscopy study of the vascular system.

Previous studies on legume pulvini suggest that the vascular system plays an important role in the redistribution of ions and transmission of stimuli during leaf's movements. However, the number of anatomical and ultrastructural studies is limited to few species. The aim of this paper is to investigate the structure and cellular features of the pulvinus vascular system of nine legume species from Brazilian cerrado, looking for structural traits pointing to its participation in the leaf's movements. Samples were excised from the medial region of opened pulvinus of Bauhinia rufa, Copaifera langsdorffii, Senna rugosa (Caesalpinioideae), Andira humilis, Dalbergia miscolobium, Zornia diphylla (Faboideae), Mimosa rixosa, Mimosa flexuosa and Stryphnodendron polyphyllum (Mimosoideae), and were prepared following light microscopy, transmission electron microscopy and histochemical standard techniques. The vascular system occupies a central position, comprises phloem and xylem and is delimited by a living sheath of septate fibers in all the species studied. This living cells sheath connects the cortex to the vascular tissues via numerous plasmodesmata. The absence of fibers and sclereids, the presence of phenolic idioblasts and the abundance and diversity of protein inclusions in the sieve tube members are remarkable features of the phloem. Pitted vessel elements, parenchyma cells with abundant cytoplasm and living fibriform elements characterize the xylem. The lack of lignified tissues and extensive symplastic continuity by plasmodesmata are remarkable features of the vascular system of pulvini of the all studied species.

Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure.

We have analysed the incorporation of [(3)H]sucrose and [(3)H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [(3)H]sucrose or [(3)H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2-3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [(3)H]Sucrose uptake was twice that of [(3)H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [(3)H]sucrose and [(3)H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [(3)H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [(3)H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [(3)H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum.

Intracellular localization of phytochrome in Robinia pseudoacacia pulvini.

The intracellular localization of phytochrome in the pulvini of Robinia pseudoacacia L. was analyzed by immunogold electron microscopy after red (R; 15 min) and far-red (FR; 5 min) irradiation 2 h after the beginning of the photoperiod. Screening of the available antibodies by immunoblotting demonstrated that none of the oat (Avena sativa L.) anti-phytochrome A (phy A) monoclonal antibodies) (MAbs) detected Robinia phytochrome. A putative Robinia phy A was detected by immunoblotting using a MAb to mustard (Sinapis alba L.) phy A (CP 2/9). No cross-reactivity was observed in blots probed with a MAb against Cucumis sativus L. phy B (mAT1). Ultrathin sections of LR White resin-embedded pulvini were immunolabelled with CP 2/9 MAb. The labelling was restricted to cortical cells and there was no evidence of labelling either in the vascular system or in the epidermis. The pattern of labelling was the same in both extensor and flexor cells irrespective of whether phytochrome was in the far-red-absorbing (Pfr) state or had reverted to the red-absorbing (Pr) form. Isolated labels and clusters of labels were randomly distributed throughout the cytoplasm. Gold particles were also found in the interior of nuclei, chloroplasts and mitochondria.

Growth dynamics and cytoskeleton organization during stem maturation and gravity-induced stem bending in Zea mays L.

Characterization of gravitropic bending in the maize stem pulvinus, a tissue that functions specifically in gravity responses, demonstrates that the pulvinus is an ideal system for studying gravitropism. Gravistimulation during the second of three developmental phases of the pulvinus induces a gradient of cell elongation across the non-growing cells of the pulvinus, with the most elongation occurring on the lower side. This cell elongation is spatially and temporally separated from normal internodal cell elongation. The three characterized growth phases in the pulvinus correspond closely to a specialized developmental sequence in which structural features typical of cells not fully matured are retained while cell maturation occurs in surrounding internodal and nodal tissue. For example, the lignification of supporting tissue and rearrangement of transverse microtubules to oblique that occur in the internode when cell elongation ceases are delayed for up to 10 d in the adjacent cells of the pulvinus, and only occurs as a pulvinus loses its capacity to respond to gravistimulation. Gravistimulation does not modify this developmental sequence. Neither wall lignification nor rearrangement of transverse microtubules occurs in the rapidly elongating lower side or non-responsive upper side of the pulvinus until the pulvinus loses the capacity to bend further. Gravistimulation does, however, lead to the formation of putative pit fields within the expanding cells of the pulvinus.

Changes in the ultrastructure of cell walls, cellulose synthesis, and glucan synthase activity from gravistimulated pulvini of oat (Avena sativa).

Ultrastructural analyses of the cell walls from top and bottom halves of gravistimulated pulvini from oat leaves show a decrease in the density of material within the cell walls from the lower halves of pulvini after 24 h of gravistimulation. Assays of cellulose synthesis with a 14C-sucrose pulse-chase experiment indicate no difference in the amount of new cellulose synthesized in top compared with bottom halves of gravistimulated pulvini. The highest rate of cellulose synthesis occurs with 12-24 h of gravistimulation. Treatment of graviresponding pulvini with 2,6-dichlorobenzonitrile (DCBN) had only a minor effect on segment gravitropic curvature. We also found that there is no difference in the activities of either glucan synthase I or glucan synthase II in top halves as compared with bottom halves of gravistimulated pulvini. We conclude that the graviresponse in oat stems is not driven by new cell wall synthesis but, rather, by changes in cell wall plasticity and osmotic potential.

Do starch statoliths act as the gravisensors in cereal grass pulvini?

To determine if starch statoliths do, in fact, act as gravisensors in cereal grass shoots, starch was removed from the starch statoliths by placing 45-day-old intact barley plants (Hordeum vulgare cv 'Larker') in the dark at 25 degrees C for 5 days. Evidence from staining with I2-KI, scanning electron microscopy, and transmission electron microscopy indicated that starch grains were no longer present in plastids in the pulvini of plants placed in the dark for 5 days. Furthermore, gravitropic curvature response in these pulvini was reduced to zero, even though pulvini from vertically oriented plants were still capable of elongating in response to applied auxin plus gibberellic acid. However, when 0.1 molar sucrose was fed to the dark pretreated, starch statolith-free pulvini during gravistimulation in the dark, they not only reformed starch grains in the starch-depleted plastids in the pulvini, but they also showed an upward bending response. Starch grain reformation appeared to precede reappearance of the graviresponse in these sucrose-fed pulvini. These results strongly support the view that starch statoliths do indeed serve as the gravisensors in cereal grass shoots.

How cereal grass shoots perceive and respond to gravity.

The leaf-sheath pulvinus of grasses presents a unique system for studying gravitropism, primarily because of its differences from other organs. The mature pulvinus is a discrete organ specialized for gravitropism: it is nongrowing in the absence of gravistimulation and capable of displaying a graviresponse independent of the rest of the plant. In this paper we present a model for gravitropism in pulvini based on recent findings from studies on the mechanisms of graviperception and graviresponse. According to this model, amyloplasts play an essential role in perceiving a change in the orientation of the pulvinus. The perception of this reorientation leads to the enhanced synthesis and release from conjugate of the auxin IAA, and the increased conjugation of gibberellin, on a localized basis. Because there is a graded growth promotion across the gravistimulated pulvinus, it is suggested that the observed hormonal asymmetry is actually an indication of a linear gradient of hormone concentration, as well as hormone response, across the pulvinus. It is further suggested that the linear gradient of hormone concentration may be predominantly the result of local changes in hormone level, rather than a product of hormonal movement into or across the pulvinus.