HELIOTROPIC LEAF MOVEMENT RESPONSE TO H+/ATPase activation,H+/ATPase inhibition,AND K+ CHANNEL INHIBITION IN VIVO

The pulvinus, located at the base of soybean leaflets, is both the light perception and motor organ for heliotropic leaf movements. Our objective was to investigate the role of plasma membrane H⁺/ATPase and TEA-sensitive K⁺ channels in mediating pulvinar response to light. The plasma membrane H+/ATPase activator, fusicoccin, plasma membrane H⁺/ATPase inhibitors, vanadate and erythrosin-B, and the K⁺ channel blocker TEA were introduced to the intact pulvinus through the transpiration stream. The pulvinus was illuminated by a vertical light beam of 1,400 μmol m^-2 s^-1 to stimulate leaf movement. Leaf orientation was measured every 5 min for 60 min of illumination. All compounds tested inhibited pulvinar bending, but concentration and uptake time required for inhibition varied: 12.5 μM fusicoccin reduced leaf movement after 3 hr uptake, 2 mM vanadate reduced leaf movement after 6 hr uptake, 100 μM erythrosin-B reduced leaf movement after 3 hr uptake, and 15 mM TEA reduced leaf movement after 6 hr uptake. In all cases final leaf angle was reduced by higher concentrations and/or increased time for uptake of the chemical into the pulvinus. Results support the hypothesis that the proximal mechanism of heliotropic movement is similar to that of nyctinastic movements.     

山东中新世山旺组紫荆属(豆科)叶化石的叶枕研究——兼论豆科植物叶枕的早期演化

提要叶枕作为叶活动的"马达器官",在豆科植物的分类、演化、发育和生理等方面都具有十分重要的研究意义,但在古植物学中似乎研究甚少。基于产自山东省临朐县中新世山旺组的叶印痕化石,结合现生紫荆叶结构和叶枕的形态观察,文中着重研究这些叶印痕化石中的叶枕形态,发现它们保存了倒卵形的上叶枕印痕和上叶枕组织腐烂分解后留下的半圆形印痕,其中一块还保存了卵形的下叶枕印痕。结合这些化石的叶结构特征,进一步确认了中新世山旺组紫荆属叶化石——华紫荆的存在,并指定一份保存于中国科学院南京地质古生物研究所的标本为后选模式,即No.39(H)。紫荆属隶属于云实亚科、紫荆族,为现存豆科植物分子系统发育树上最基部的类群之一,因此该属的化石历史对认识豆科植物的系统发育、早期演化和分异以及生物地理历史都至关重要。然而,由于过去缺乏详细的叶结构和叶枕研究,大多数以往报道的产自欧亚大陆和北美新生代的紫荆属叶印痕化石种都受到质疑、否定或亟需进一步研究。迄今为止,紫荆属最早的、具叶枕的叶化石发现于美国俄勒冈州晚始新世的约翰迪组,而最早的豆科叶枕则发现于阿根廷巴塔哥尼亚早古新世萨拉曼卡组中的一种可能属于含羞草亚科植物的小叶中。结合近来的比较形态解剖学研究结果,作者赞同先前的假说——紫荆属的单叶和叶枕类型实质上都是衍生的性状,而最原始的豆科植物长着羽状复叶,其复叶叶柄和小叶叶柄上可能都有各自的叶枕。     

Compartmentation of soluble carbohydrates,of starch and of malate in motor organs (pulvini) and other parts of Phaseolus coccineus L. leaves

Quantitative histochemistry was used to investigate the tissue-specific compartmentation of soluble carbohydrates (sucrose, glucose, fructose), starch and malate in the laminar pulvinus, leaf blade and petiole of Phaselous coccineus L. at day and night positions of diurnal leaf movement. Total carbohydrate levels measured in a series of cross sections along individual pulvini of 24-d-old plants showed only small differences between the day and night positions of the respective leaf. In contrast, the level of malate changed during diurnal leaf movement, especially in the central part of a pulvinus. The levels of glucose and fructose in the pulvinus increased towards the transition zones between the pulvinus and lamina, and pulvinus and petiole, and this trend was even more pronounced for starch. By contrast, sucrose levels were highest in the pulvinus proper. The transverse compartmentation of metabolites was studied in distinct, approx. 0.5-mm-thick tissue slices from the central part of a pulvinus. These were dissected further into up to 14 distinct subsamples (bundle, bundle sheath, motor tissues, flanks). Irrespective of the position of the leaf (day or night), the central vascular core and the surrounding bundle sheath had high levels of sucrose (up to 500 mmol-(kg DW)^–1) and low levels of glucose and fructose (below 100 mmol-(kg DW)^–1), while in the cortex the situation was reversed. In the night position the level of sucrose decreased by approx. 30% in the bundle sheath and the central vascular core but not in the other sections. We thus suggest that because of the relatively small diurnal changes in their cortical pools, soluble sugars are not involved in the osmotic processes resulting in leaf movement. In contrast, pulvini from 14-d-old plants showed an interesting diurnal change in starch and malate pools in the outermost layer of the extensor. Here starch increased at night while the malate pool was lowered nearly stoichiometrically. Inverse pool sizes were found in the day position of the respective leaves. Although less significant, the opposite diurnal variation occurred in samples taken from the flexor region. We thus were able to locate areas of different carbohydrate activities in the laminar pulvinus of P. coccineus. The central vascular core, including the bundle sheath, is involved in temporary storage of photoassimilates, and the cortical regions are responsible for osmotically driven leaf movement. The results are discussed with respect to guard-cell physiology.Abbreviations CLP cut-leaf pulvini - ILP intact-leaf pulvini This work was supported by a grant from the Deutsche Forschungsgemeinschaft.     

SECONDARY PULVINUS OF ROBINIA PSEUDOACACIA (LEGUMINOSAE): STRUCTURAL AND ULTRASTRUCTURAL FEATURES

The structure of the secondary pulvinus of Robinia pseudoacacia has been examined together with ultrastructural features of motor cells both in open and closed pulvini, to identify ultrastructural changes associated with leaflet movement. Pulvini have a central vascular core bordered by thick-walled collenchyma cells, which in turn are surrounded by several layers of cortical parenchyma cells. Cortical motor cells exhibit ultrastructural features similar to those reported in homologous cells of other pulvini. The vacuolar compartment contains two kinds of vacuoles: nontannin vacuoles, which change both in number and size during leaflet movement, and tannin vacuoles, which may act as an ion reservoir. No differences in wall thickness were found between flexor and extensor motor cells. Thick walls of collenchyma cells show numerous pits with plasmodesmata through which the phloem parenchyma cells and the inner cortical motor cells are connected. Tannin vacuoles and calcium oxalate crystals are common inclusions of phloem parenchyma cells. The tissue arrangement and the occurrence of pits with plasmodesmata in the central cylinder cells provide evidence of symplastic continuity through the central cylinder between the extensor and flexor regions of the motor organs. The greater amplitude of Robinia leaflet movements may be related to the extension of motor regions, the scarcity of lignification in the central vascular core, and the thin flexor walls.     

The Role of the Lens in Controlling Heat-induced Breakdown of Testa-imposed Dormancy in Native Australian Legumes

A high proportion of seeds of most species of the Fabaceae havetesta-imposed dormancy, which prevents them from imbibing watereven under favourable environmental conditions. This allowsseeds to accumulate in a persistent soil seed bank. Dormancyis usually considered to be broken only when the testa is disrupted,particularly at the lens. In Mediterranean-type ecosystems,the primary natural mechanism causing the breakdown of thisdormancy is the disruption of testas in the soil seed bank byheat from fires that warms the soil in which the seed bank occurs.We examined experimentally the role of the lens in controllingthis heat-induced breakdown of dormancy in 16 native Australianspecies of the Faboideae and Mimosoideae. Two groups of patternsare recognized: species of the Mimosoideae: Acacieae and Faboideae:Mirbelieae, where water permeability is regulated by the lens;and species of the Faboideae: Bossiaeeae, where water permeabilityapparently occurs at non-localized regions of the testa. Thespecies in these two groups differ in the structure of theirtestas. Thus, testa-imposed dormancy does not represent a singledormancy mechanism in legumes, as is often assumed when dormancyis broken artificially.Copyright 1998 Annals of Botany Company Leguminosae, Fabaceae, seed dormancy, hard-seededness, germination, lens, strophiole, Australia.     

Crystals in tapetal cells of the Leguminosae

In a survey of the Leguminosae (167 species of 89 genera), calcium oxalate crystals were found in tapetal cells in all three subfamilies (84 species of 52 genera). Crystal form ranged from small scattered crystals to lens-like to various large prismatic types, and included the first report of styloid crystals in the Caesalpinioideae. Over half of the caesalpinioid and papilionoid legumes examined had crystals. Crystals were not common in the Mimosoideae, however, but this may be because of the small sample. The distribution of species with various crystal types, and of species lacking crystals, did not reveal any patterns that conform to the presently defined taxa. Crystals in tapetal cells have previously been reported only in the Commelinaceae and Labiatae.     

Extensor and flexor protoplasts from samanea pulvini : I. Isolation and initial characterization

Protoplasts were isolated from extensor and flexor regions of open pulvini of the nyctinastic tree Samanea saman. Both types of protoplasts undergo many changes during isolation. Extensor protoplasts are univacuolate in vivo, but some become multivacuolate. All flexor protoplasts are univacuolate. In an open pulvinus, extensor cells have a higher osmotic pressure than flexor cells. However, both types of protoplasts can be isolated with optimal yield using the same osmoticum (0.5 molar sorbitol) in the digestion medium. This suggests that some leakage of osmoticum occurs during harvest or digestion, especially from extensor tissue. Despite these changes, both types of protoplasts extrude protons in response to 10 micromolar fusicoccin (1.6-1.8 nanoequivalent/10⁶ protoplasts/minute), demonstrating that the protoplasts are metabolically active and that proton transport mechanisms must be at least partially functional. The changes in vacuolar structure and osmotic pressure are what one might expect if the protoplasts, which are isolated from open pulvini, take on characteristics of cells in a closed pulvinus.     

Effects of glycine on dark-and ligh-induced pulvinar movements and modifications of proton fluxes in the pulvinus of Mimosa pudica during glycine uptake

Glycine (1–50 mM) increases the rate of the dark-induced (scotonastic) movements and decreases the amplitude and the rate of the light-induced (photonastic) movements of the secondary pulvini of Mimosa pudica leaves. The uptake of glycine is accompanied by a long-lasting dose-dependent increase in the alkalinity of the bathing medium of the excised pulvini. The data are in agreement with a H⁺-glycine co-transport mechanism within the pulvinar cells. Fusicoccin (50 M), known to promote H⁺–K⁺ exchange, antagonizes the effects of glycine on the movements and the alkalization of the bathing medium of the excised pulvini. The present results argue for the hypothesis that proton fluxes mediate the scotonastic and photonastic pulvinar movements.Abbreviations Gly glycine - FC fusicoccin - P₁ primary pulvinus - P₂ secondary pulvinus     

Histochemical Localization of Calcium Oxalate Crystals in Starch Grains of Yams (Dioscorea)

The bulbils and/or tubers of seven species of yams (Dioscorea)were examined for crystal content using light microscopy andhistochemistry. Calcium oxalate crystals in the form of raphide bundles werelocalized in the parenchymatous tissues. Within starch grains,crystals of various shapes and sizes were observed. The variationin shape and sizes of the intra-amylar crystals could be exploitedfor taxonomic purposes. Calcium oxalate crystals appear to serve a storage functionin these starch grains. Yams, Dioscorea, raphides, oxalate crystals, histochemistry     

Paper chromatographic survey of anthocyanins in leguminosae

Flower anthocyanins of 22 leguminous species, of which 20 species belong to the subfamily Faboideae, were examined. In the present study, 21 kinds of anthocyanin were found and their distribution pattern in 22 species was discussed.Albizia julibrissin belonging to the subfamily Mimosoideae contained only cyanidin 3-glucoside, which was quite different from the pigment constitutents in other species. Anthocyanins ofCercis chinensis belonging to the subfamily Caesalpinioideae were similar to those of species of the subfamily Faboideae. Malvidin glycoside was contained as a main pigment in 14 species examined. Malvidin and petunidin glycosides were most frequent and occurred in 20 legumes. Delphinidin glycoside, cyanidin glycoside and peonidin glycoside were present in descending order.     

On the role of tannin vacuoles in several nastic leaf responses

Summary Experiments were done to relate the presence of tannin vacuoles to plant movements. When surveyed, 4 out of 10 species exhibited rapid thigmonastic or nyctinastic movements, and only those 4 species had tannin vacuoles in their motor cells. Interestingly, plants ofAlbizzia julibrissin whose seed was obtained in New Haven, Connecticut had rapid nyctinasty and tannin vacuoles in the tertiary pulvini, whereasAlbizzia collected in Winston-Salem, North Carolina had neither. Nyctinasty ofMimosa pudica andAlbizzia NH, both of which had tannin vacuoles, was at the rate of 4.2 °/min, whereasAlbizzia WS andSamanea saman, neither of which had tannin vacuoles, exhibited leaflet closure at the rate of 2.0–2. 1 °/min. Pretreatment with the Ca²⁺ channel agonist Bay K-8644 increased, and with the antagonist verapamil decreased, the rate of leaflet closure only inMimosa andAlbizzia NH. Pretreatment with the Ca²⁺ inhibitor Erythrosin B substantially blocked reopening only inMimosa andAlbizzia NH. We conclude that tannin vacuoles assist nastic movements to be rapid by acting as a store of Ca²⁺ and releasing it as a second messenger upon mechanical perturbation or darkness.Abbreviations FR far-red irradiation - MP mechanical perturbation - TV tannin vacuoles     

<Emphasis Type="Italic">SLEEPLESS</Emphasis>, a gene conferring nyctinastic movement in legume

A genetic approach was attempted to identify the gene responsible for nyctinastic movement in legume. Seeds of the model legume Lotus japonicus were treated with ethylmethane sulfonate and screening of 40,000 M2 seeds led to the isolation of one mutant named sleepless. sleepless is incapable of closing its leaflets towards the adaxial side at night. The pulvini at the leaflet base were found to be replaced with petiole-like structure in sleepless. Wild-type pulvini comprise many compressed cells, whereas the corresponding region in sleepless is made up of roundish cells in the cortical parenchyma and highly elongated cells in the epidermis, particularly in the leaf-length direction. Based on the results of histological examination, I propose a possible model of a developmental pathway leading to nyctinastic movement. Electronic Publication     

Mechanical and electrical anisotropy in Mimosa pudica pulvini

Thigmonastic or seismonastic movements in Mimosa pudica, such as the response to touch, appear to be regulated by electrical, hydrodynamical and chemical signal transduction. The pulvinus of Mimosa pudica shows elastic properties, and we found that electrically or mechanically induced movements of the petiole were accompanied by a change of the pulvinus shape. As the petiole falls, the volume of the lower part of the pulvinus decreases and the volume of the upper part increases due to the redistribution of water between the upper and lower parts of the pulvinus. This hydroelastic process is reversible. During the relaxation of the petiole, the volume of the lower part of the pulvinus increases and the volume of the upper part decreases. Redistribution of ions between the upper and lower parts of a pulvinus causes fast transport of water through aquaporins and causes a fast change in the volume of the motor cells. Here, the biologically closed electrochemical circuits in electrically and mechanically anisotropic pulvini of Mimosa pudica are analyzed using the charged capacitor method for electrostimulation at different voltages. Changing the polarity of electrodes leads to a strong rectification effect in a pulvinus and to different kinetics of a capacitor discharge if the applied initial voltage is 0.5 V or higher. The electrical properties of Mimosa pudica''s pulvini were investigated and the equivalent electrical circuit within the pulvinus was proposed to explain the experimental data. The detailed mechanism of seismonastic movements in Mimosa pudica is discussed.Key words: electrophysiology, plant electrostimulation, pulvinus, Mimosa pudica, charged capacitor method, electrical circuits, ion channels     

Osmoregulation of leaf motor cells

"Osmotic Motors"--the best-documented explanation for plant leaf movements--frequently reside in specialized motor leaf organs, pulvini. The movements result from dissimilar volume and turgor changes in two oppositely positioned parts of the pulvinus. This Osmotic Motor is powered by a plasma membrane proton ATPase, which drives KCl fluxes and, consequently, water, across the pulvinus into swelling cells and out of shrinking cells. Light signals and signals from the endogenous biological clock converge on the channels through which these fluxes occur. These channels and their regulatory pathways in the pulvinus are the topic of this review.     

Brassinosteroid homeostasis is critical for the functionality of the Medicago truncatula pulvinus

Many plant species open their leaves during the daytime and close them at night as if sleeping. This leaf movement is known as nyctinasty, a unique and intriguing phenomenon that been of great interest to scientists for centuries. Nyctinastic leaf movement occurs widely in leguminous plants, and is generated by a specialized motor organ, the pulvinus. Although a key determinant of pulvinus development, PETIOLULE-LIKE PULVINUS (PLP), has been identified, the molecular genetic basis for pulvinus function is largely unknown. Here, through an analysis of knockout mutants in barrelclover (Medicago truncatula), we showed that neither altering brassinosteroid (BR) content nor blocking BR signal perception affected pulvinus determination. However, BR homeostasis did influence nyctinastic leaf movement. BR activity in the pulvinus is regulated by a BR-inactivating gene PHYB ACTIVATION TAGGED SUPPRESSOR1 (BAS1), which is directly activated by PLP. A comparative analysis between M. truncatula and the non-pulvinus forming species Arabidopsis and tomato (Solanum lycopersicum) revealed that PLP may act as a factor that associates with unknown regulators in pulvinus determination in M. truncatula. Apart from exposing the involvement of BR in the functionality of the pulvinus, these results have provided insights into whether gene functions among species are general or specialized.

Nyctinasty is triggered by the pulvinus, a motor organ located at the base of the leaf and brassinosteroids is involved in functionality of pulvinus for leaf movement.     

STRUCTURE OF GRAVITY-SENSITIVE SHEATH AND INTERNODAL PULVINI IN GRASS SHOOTS

The location and some morphological, anatomical, and functional aspects of the gravity-sensitive pulvini of a selected number of grass shoots are examined. There are two distinct gravity-sensitive regions near the nodal regions of Gramineae. One, the leaf sheath pulvinus, is located at the base of the sheathing leaf bases, and is characteristic of the subfamily Festucoideae. The other, the internodal pulvinus, is located at the base of the internode, a little above the nodal joint. Most members of the Panicoideae possess internodal pulvini, in addition to more or less developed leaf sheath pulvini. Three members of the Oryzoideae examined possess leaf sheath pulvini only, while Phragmites australis (Arundinoideae) possesses both leaf sheath and internodal pulvini. Leaf sheath pulvini of some grasses develop hairs, cork-silica cell pairs or stomatal apparatuses over the epidermis while many others are devoid of any such idioblasts. Both the leaf sheath and internodal pulvini of all grasses examined preferentially exclude, or accumulate very little silica, whereas the regions of the shoot immediately above and below the pulvini in these same grasses accumulate large quantities of silica. Pulvini remain unsilicified or poorly silicified throughout their life and even after several days following geotropic bending. Pulvini are also distinguished from the regions above and below them by the lack of lignin in the bundle cap cells. Lignin is found only in the xylem vascular tissue, and this consists of annular and helical vessel elements only. The bundle cap cells are rich in pectin and are described as collenchymatous. All pulvini possess specialized zones of cells containing starch statoliths. In response to horizontal displacement of the shoots, the lower side of the pulvini grows by cell elongation only. The collenchymatous cells stretch in a manner that results in alternately thin and thick regions of cell wall.     

Diverse patterns of cell wall mannan/galactomannan occurrence in seeds of the Leguminosae

Endosperms from seeds of different subfamilies of Leguminosae were submitted to sequential aqueous and alkaline aqueous extractions. The extractions from species belonging to the Mimosoideae and Faboideae subfamilies yielded galactomannans with constant Man:Gal ratios, whereas the extractions from Caesalpinioideae seeds gave rise to galactomannans with increasing values of the Man:Gal ratio. The presence of a family of galactomannans within the same species may be a trait found only in Caesalpinioideae subfamily. The final insoluble residues that were obtained after the removal of galactomannans from the Caesalpinioideae and Faboideae subfamilies are composed of pure mannans and do not contain cellulose, while those from the Mimosoideae subfamily are composed of cellulose. A mannan was isolated from the unripe endosperm of Caesalpinia pulcherrima, suggesting no developmental relationship between galactomannan and mannan. These results are consistent with the presence of a distinctive cell wall pattern in the endosperms of Leguminosae species.     

Elemental analysis of freeze-dried thin sections of Samanea motor organs: barriers to ion diffusion through the apoplast

Leaflet movements in the legume Samanea saman are dependent upon massive redistribution of potassium (K), chloride (Cl), and other solutes between opposing (extensor and flexor) halves of the motor organ (pulvinus). Solutes are known to diffuse through the apoplast during redistribution. To test the possibility that solute diffusion might be restricted by apoplastic barriers, we analyzed elements in the apoplast in freeze-dried cryosections of pulvini using scanning electron microscopy/x-ray microanalysis. Large discontinuities in apoplastic K and Cl at the extensor-flexor interface provide evidence for a barrier to solute diffusion. The barrier extends from the epidermis on upper and lower sides of the pulvinus to cambial cells in the central vascular core. It is completed by hydrophobic regions between phloem and cambium, and between xylem rays and surrounding vascular tissue, as deduced by discontinuities in apoplastic solutes and by staining of fresh sections with lipid-soluble Sudan dyes. Thus, symplastic pathways are necessary for ion redistribution in the Samanea pulvinus during leaflet movement. In pulvini from leaflets in the closed state, all cells on the flexor side of the barrier have high internal as well as external K and Cl, whereas cells on the extensor side have barely detectable internal or external K or Cl. Approximately 60% of these ions are known to migrate to the extensor during opening; all return to the flexor during subsequent closure. We propose that solutes lost from shrinking cells in the outer cortex diffuse through the apoplast to plasmodesmata-rich cells of the inner cortex, collenchyma, and phloem; and that solutes cross the barrier by moving through plasmodesmata.     

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°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.