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.

Reactive oxygen species regulate leaf pulvinus abscission zone cell separation in response to water-deficit stress in cassava.

Cassava (Manihot esculenta Crantz) plant resists water-deficit stress by shedding leaves leading to adaptive water-deficit condition. Transcriptomic, physiological, cellular, molecular, metabolic, and transgenic methods were used to study the mechanism of cassava abscission zone (AZ) cell separation under water-deficit stress. Microscopic observation indicated that AZ cell separation initiated at the later stages during water-deficit stress. Transcriptome profiling of AZ suggested that differential expression genes of AZ under stress mainly participate in reactive oxygen species (ROS) pathway. The key genes involved in hydrogen peroxide biosynthesis and metabolism showed significantly higher expression levels in AZ than non-separating tissues adjacent to the AZ under stress. Significantly higher levels of hydrogen peroxide correlated with hydrogen peroxide biosynthesis related genes and AZ cell separation was detected by microscopic observation, colorimetric detection and GC-MS analyses under stress. Co-overexpression of the ROS-scavenging proteins SOD and CAT1 in cassava decreased the levels of hydrogen peroxide in AZ under water-deficit stress. The cell separation of the pulvinus AZ also delayed in co-overexpression of the ROS-scavenging proteins SOD and CAT1 plants both in vitro and at the plant level. Together, the results indicated that ROS play an important regulatory role in the process of cassava leaf abscission under water-deficit stress.

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.

Anoxia promotes gravitropic curvature in rice pulvini but inhibits it in wheat and oat pulvini.

Gravitropic curvature of pulvini of wheat and oat stem segments gradually declined with decreasing atmospheric O2 concentration and was almost completely blocked under anoxia, whereas that of rice stem segments was enhanced under hypoxia and anoxia. Anoxia substantially increased the ethanol content in pulvini of gravistimulated stem segments in rice, wheat and oat, but the ethanol content showed no marked difference between rice pulvini and wheat and oat pulvini. The concentrations of exogenous ethanol and acetaldehyde required to inhibit the gravitropic curvature of pulvini were significantly higher in rice segments than in wheat and oat segments. However, in all three species, the concentrations of ethanol and acetaldehyde required to completely inhibit curvature were several-fold higher than the endogenous levels that accumulated in pulvini gravistimulated in N2. The pulvini of rice segments gravistimulated in N2 did not contain much more ATP than those of wheat or oat segments gravistimulated in N2. When applied unilaterally to the pulvini of vertically oriented stem segments incubated in N2, indole-3-acetic acid induced bending in rice stem segments but not in wheat and oat stem segments. Transference of graviresponsive pulvini of rice, as well as those of wheat and oat, from aerobic conditions to anaerobic conditions led to cessation of gravitropic curvature within several minutes, but subsequently only gravitropic curvature of anoxic rice pulvini was completely recovered within 2 h. A large portion of this recovery was blocked by cordycepin, a transcription inhibitor. These results suggested that anoxia-induced expression of any gene or genes enables rice pulvini to respond to gravistimulation under anaerobic conditions, and that such a gene or genes might be unrelated to ethanol fermentation and ATP production in anaerobic conditions.

Identification and characterization of petiolule-like pulvinus mutants with abolished nyctinastic leaf movement in the model legume Medicago truncatula.

Leaves of many plant species open during the day and fold at night. Diurnal leaf movement, named nyctinasty, has been of great interest to researchers since Darwin's time. Nyctinastic leaf movement is generated by the pulvinus, which is a specialized motor organ located at the base of leaf and leaflet. The molecular basis and functional reason behind nyctinasty are unknown. In a forward screening of a retrotransposon-tagged mutant population of Medicago truncatula, four petiolule-like pulvinus (plp) mutant lines with defects in leaf movement were identified and characterized. Loss of function of PLP results in the change of pulvini to petiolules. PLP is specifically expressed in the pulvinus, as demonstrated by quantitative reverse-transcription polymerase chain reaction analysis, expression analysis of a PLP promoter-ß-glucuronidase construct in transgenic plants and in situ hybridization. Microarray analysis revealed that the expression levels of many genes were altered in the mutant during the day and at night. Crosses between the plp mutant and several leaf pattern mutants showed that the developmental mechanisms of pulvini and leaf patterns are likely independent. Our results demonstrated that PLP plays a crucial role in the determination of pulvinus development. Leaf movement generated by pulvini may have an impact on plant vegetative growth.

TCP transcription factor, BRANCH ANGLE DEFECTIVE 1 (BAD1), is required for normal tassel branch angle formation in maize.

In grass inflorescences, a structure called the "pulvinus" is found between the inflorescence main stem and lateral branches. The size of the pulvinus affects the angle of the lateral branches that emerge from the main axis and therefore has a large impact on inflorescence architecture. Through EMS mutagenesis we have identified three complementation groups of recessive mutants in maize having defects in pulvinus formation. All mutants showed extremely acute tassel branch angles accompanied by a significant reduction in the size of the pulvinus compared with normal plants. Two of the complementation groups correspond to mutations in the previously identified genes, RAMOSA2 (RA2) and LIGULELESS1 (LG1). Mutants corresponding to a third group were cloned using mapped-based approaches and found to encode a new member of the plant-specific TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR) family of DNA-binding proteins, BRANCH ANGLE DEFECTIVE 1 (BAD1). BAD1 is expressed in the developing pulvinus as well as in other developing tissues, including the tassels and juvenile leaves. Both molecular and genetics studies show that RA2 is upstream of BAD1, whereas LG1 may function in a separate pathway. Our findings demonstrate that BAD1 is a TCP class II gene that functions to promote cell proliferation in a lateral organ, the pulvinus, and influences inflorescence architecture by impacting the angle of lateral branch emergence.

Effects of staurosporine, okadaic acid and sodium fluoride on protein phosphorylation in graviresponding oat shoot pulvini.

This study focuses on the characterization of protein phosphorylation in the gravitropic response in oat shoot pulvini through the use of inhibitors of this process, namely staurosporine, okadaic acid and sodium fluoride. These three inhibitors reduce gravitropic curvature and cause changes in the phosphorylation of 38- and 50-kDa soluble proteins which show different levels of phosphorylation between lower and upper halves of gravistimulated pulvini. A kinetic analysis of phosphorylation shows that the 38- and 50-kDa soluble proteins exhibit different levels of phosphorylation between lower and upper halves of graviresponsive pulvini at 5 min after initiation of gravistimulation of stems. In addition, the phosphorylation of 63- and 70-kDa proteins from a total membrane preparation increases in lower halves of the pulvini following gravistimulation. These phosphoproteins are not found in the plasma membrane fraction. Taken together, at least four kinds of phosphoproteins are gravi-related. Of these, the 38- and 50-kDa soluble phosphoproteins may be involved in the regulation of early stages of the gravitropic response.

Transient and sustained increases in inositol 1,4,5-trisphosphate precede the differential growth response in gravistimulated maize pulvini.

The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. We observed a transient 5-fold increase in inositol 1,4,5-trisphosphate (IP3) within 10 s of gravistimulation in the lower half of the pulvinus, indicating that the positional change was sensed immediately. Over the first 30 min, rapid IP3 fluctuations were observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently had higher IP3, and IP3 levels on the lower side continued to increase up to approximately 5-fold over basal levels before visible growth. As bending became visible after 8-10 h, IP3 levels returned to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increased transiently within 10 min of gravistimulation, suggesting that the increased IP3 production was accompanied by an up-regulation of phosphatidylinositol 4, 5-bisphosphate biosynthesis. Neither IP3 levels nor phosphatidylinositol 4-phosphate 5-kinase activity changed in pulvini halves from vertical control plants. Our data indicate the involvement of IP3 and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP3 provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response.

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.

Hormonal and gravitropic specificity in the regulation of growth and cell wall synthesis in pulvini and internodes from shoots of Avena sativa L. (oat).

Segments can be cut from the peduncular-1 internode of oat (Avena sativa L.) shoots so as to contain the graviresponsive leaf-sheath pulvinus and gibberellin-sensitive internodal tissue. Incorporation of [14C]glucose was used to monitor cell wall synthesis in these two tissues as affected by gravistimulus, indoleacetic acid (IAA), gibberellic acid (GA3), and fusicoccin (FC). Pulvinar cell wall synthesis was promoted by IAA and FC (both within about 1 h), as well as by gravistimulus (starting between 3 and 6 h), whereas GA3 had no effect on nongravistimulated pulvini. In contrast, GA3 and FC promoted internodal cell wall synthesis (initiated between 1 and 2 h), whereas IAA and gravistimulus caused a decrease in internodal uptake. FC preferentially promoted incorporation into the matrix component of the wall in both tissues. Gravistimulus failed to increase responsiveness of pulvinar tissue to IAA, whereas GA3 partially overcame gravistimulus-promoted incorporation into pulvinar cell wall, probably because of preferential movement of label into the rapidly elongating internode. The results demonstrate that these eight stimulus/tissue combinations can be examined easily in an isolated 10-mm stem segment, providing new opportunities for the comparative study of tissue- and stimulus-specific events in gene regulation and signal transduction in agronomically important cereals.

The role of calcium in growth induced by indole-3-acetic acid and gravity in the leaf-sheath pulvinus of oat (Avena sativa).

Leaf-sheath pulvini of excised segments from oat (Avena sativa L.) were induced to grow by treatment with 10 micromoles indole-3-acetic acid (IAA), gravistimulation, or both, and the effects of calcium, EGTA, and calcium channel blockers on growth were evaluated. Unilaterally applied calcium (10 mM CaCl2) significantly inhibited IAA-induced growth in upright pulvini but had no effect on growth induced by either gravity or gravity plus IAA. Calcium alone had no effect on upright pulvini. The calcium chelator EGTA alone (10 mM) stimulated growth in upright pulvini. However, EGTA had no effect on either IAA- or gravity-induced growth but slightly diminished growth in IAA-treated gravistimulated pulvini. The calcium channel blockers lanthanum chloride (25 mM), verapamil (2.5 mM), and nifedipine (2.5 mM) greatly inhibited growth as induced by IAA (> or = 50% inhibition) or IAA plus gravity (20% inhibition) but had no effect on gravistimulated pulvini. Combinations of channel blockers were similar in effect on IAA action as individual blockers. Since neither calcium ions nor EGTA significantly affected the graviresponse of pulvini, we conclude that apoplastic calcium is unimportant in leaf-sheath pulvinus gravitropism. The observation that calcium ions and calcium channel blockers inhibit IAA-induced growth, but have no effect on gravistimulated pulvini, further supports previous observations that gravistimulation alters the responsiveness of pulvini to IAA.

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.

Dynamics of auxin movement in the gravistimulated leaf-sheath pulvinus of oat (Avena sativa).

The role of auxin redistribution in the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa L.) was assessed using 3H-indole-3-acetic acid (3H-IAA) preloaded into isolated pulvini. When pulvini were totally isolated from subtending nodal tissue as well as leaf-sheath and internode, gravistimulation failed to induce an asymmetric growth response. Presence of either the nodal tissue or the internode/leaf-sheath tissue was sufficient to restore a normal graviresponse. Gravistimulation of totally isolated pulvini inhibited basipetal export of label (i.e., 3H-IAA) without generating any asymmetry of label within the pulvinus. In contrast, gravistimulation of pulvini with nodes intact generated an asymmetric distribution of label (initiation by 1 h; final ratio, lower/upper = 1.5) as well as the upward bending response. The kinetics of formation of the asymmetry of label paralleled the kinetics of initiation of the asymmetric growth response. The addition of 0.1 M sucrose to all agar blocks shortened both the time to initiation of label redistribution and the time to initial upward bending. However, sucrose did not change the final magnitude of label asymmetry although it increased the final steady state bending rate four fold. The inhibitors of polar auxin transport N-1-naphthylphthalamaic acid (NPA), 2,3,5-triiodobenzoic acid (TIBA), morphactin, naringenin, kaempferol and myricetin all significantly decreased the bending response of oat pulvini, but this inhibition was less than 50%. In contrast, TIBA and naringenin (each at 100 micromoles), effectively eliminated the redistribution of label, but did not eliminate the bending response. These results indicate that the active basipetal export of auxin is inhibited by gravistimulation of the oat pulvinus, while active lateral transport is induced. It is concluded that, while lateral transport of auxin occurs following gravistimulation, it is not necessary for a graviresponse. Other processes, such as localized changes in tissue responsiveness or the conversion of conjugated hormone to free (active) hormone, may suffice to drive the graviresponse.

Cell wall and enzyme changes during the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa).

The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response accompanied by several asymmetric processes, including degradation of starch and cell wall synthesis. To understand further the cellular and biochemical events associated with the graviresponse, changes in cell walls and their constituents and the activities of related enzymes were investigated in excised pulvini. Asymmetric increases in dry weight with relatively symmetric increases in wall weight accompanied the graviresponse. Starch degradation could not account for increases in wall weight. However, a strong asymmetry in invertase activity indicated that hydrolysis of exogenous sucrose could contribute significantly to the increases in wall and dry weights. Most cell wall components increased proportionately during the graviresponse. However, beta-D-glucan did not increase symmetrically, but rather increased in proportion in lower halves of gravistimulated pulvini. This change resulted from an increase in glucan synthase activity in lower halves. The asymmetry of beta-D-glucan content arose too slowly to account for initiation of the graviresponse. A similar pattern in change in wall extensibility was also observed. Since beta-D-glucan was the only wall component to change, it is hypothesized that this change is the basis for the change in wall extensibility. Since wall extensibility changed too slowly to account for growth initiation, it is postulated that asymmetric changes in osmotic solutes act as the driving factor for growth promotion in the graviresponse, while wall extensibility acts as a limiting factor during growth.

Plant movements caused by differential growth--unity or diversity of mechanisms?

A very wide range of plant organ movements have been described and yet it is not clear how each of them is related to the others. This uncertainty has had two undesirable consequences. Firstly, some workers have accepted the idea of a vague unity in the area and have subsequently been misled by information obtained in one system and applied without adequate justification to a study of a quite different system. Secondly, some researchers have evoked a possible diversity to explain why a particular mechanistic explanation may continue to be valid even when the model fails to explain events of a very similar nature in a slightly different system. We argue that this confusion has resulted from a classification of organ movements which has been based on functional rather than on mechanistic considerations. Mechanistic unity is to be expected on evolutionary grounds. This unity, however, may apply only to certain elements of the stimulus-response chain, at certain levels of organization. It follows from this that in seeking this unity, comparisons should be made between equivalent elements of the stimulus-response chain at the same level of organization in different systems. Only when this is done will theories built around the concept of unity provoke meaningful discussion.

Competency for graviresponse in the leaf-sheath pulvinus of Avena sativa: onset to loss.

The development of the leaf-sheath pulvinus of oat (Avena sativa L. cv. Victory) was studied in terms of its competency to respond to gravistimulation. Stages of onset of competency, maximum competency and loss of competency were identified, using the length of the supertending internode as a developmental marker. During the early phases in the onset of competency, the latency period between stimulus and graviresponse decreased and the steady state response rate increased significantly. When fully competent, the latency period remained constant as the plant continued to develop, suggesting that the latency period is relatively insensitive to quantitative changes (e.g., in carbohydrate or nutrient availability) at the cell level within the plant. In contrast, the response rate was found to increase with plant development, indicating that graviresponse rate is more strongly influenced by quantitative cellular changes. The total possible graviresponse of a single oat pulvinus was confirmed to be significantly less than the original presentation angle. This was shown to not result from a loss of competency, since the graviresponse could be reinitiated by increasing the presentation angle. As a result of the low overall graviresponse of individual pulvini, two or more pulvini are required to bring the plant apex to the vertical. This was determined to occur though the sequential, rather than simultaneous, action of successive pulvini, since a given pulvinus lost competency to gravirespond shortly after the next pulvinus became fully competent.

Effect of dark pretreatment on the kinetics of response of barley pulvini to gravistimulation and hormones.

Starch in pulvinus amyloplasts of barley (Hordeum vulgare cv Larker) disappears when 45-day-old, light-grown plants are given 5 days of continuous darkness. The effect of this loss on the pulvinus graviresponse was evaluated by following changes in the kinetics of response during the 5-day dark period. Over 5 days of dark pretreatment, the lag to initial graviresponse and the subsequent half-time to maximum steady state bending rate increased significantly while the maximum bending rate did not change. The change in response to applied indoleacetic acid (100 micromolar) plus gibberellic acid (10 micromolar) without gravistimulation, under identical dark pretreatments, was used as a model system for the response component of gravitropism. Dark pretreatment did not change the lag to initial response following hormone application to vertical pulvini, but both the maximum bending rate and the half-time to the maximum rate were significantly reduced. Also, after dark pretreatment, significant bending responses following hormone application were observed in vertical segments with or without added sucrose, while gravistimulation produced a response only if segments were given sucrose. These results indicate that starch-filled amyloplasts are required for the graviresponse of barley pulvini and suggest that they function in the stimulus perception and signal transduction components of gravitropism.

Altered growth response to exogenous auxin and gibberellic acid by gravistimulation in pulvini of Avena sativa.

Pulvini of excised segments from oats (Avena sativa L. cv Victory) were treated unilaterally with indoleacetic acid (IAA) or gibberellic acid (GA3) with or without gravistimulation to assess the effect of gravistimulation on hormone action. Optimum pulvinus elongation growth (millimeters) and segment curvature (degrees) over 24 hours were produced by 100 micromolar IAA in vertical segments. The curvature response to IAA at levels greater than 100 micromolar, applied to the lower sides of gravistimulated (90 degrees) pulvini, was significantly less than the response to identical levels in vertical segments. Furthermore, the bending response of pulvini to 100 micromolar IAA did not vary significantly over a range of presentation angles between 0 and 90 degrees. In contrast, the response to IAA at levels less than 10 micromolar, with gravistimulation, was approximately the sum of the responses to gravistimulation alone and to IAA without gravistimulation. This was observed over a range of presentation angles. Also, GA3 (0.3-30 micromolar) applied to the lower sides of horizontal segments significantly enhanced pulvinus growth and segment curvature, although exogenous GA3 over a range of concentrations had no effect on pulvinus elongation growth or segment curvature in vertical segments. The response to GA3 (10 micromolar) plus IAA (1.0 or 100 micromolar) was additive for either vertical or horizontal segments. These results indicate that gravistimulation produces changes in pulvinus responsiveness to both IAA and GA3 and that the changes are unique for each growth regulator. It is suggested that the changes in responsiveness may result from processes at the cellular level other than changes in hormonal sensitivity.

Investigations into the possible regulation of negative gravitropic curvature in intact Avena sativa plants and in isolated stem segments by ethylene and gibberellins.

Using Avena sativa L. cv. Victory oat seedlings and excised p-1 stem segments (including the p-1 and p-2 internodes) the effect of exogenously supplied ethylene and the removal of ethylene on internodal extension and gravitropic bending was assessed. Similarly, the ability of the excised system to respond to gravistimulation was assessed in the presence of inhibitors of ethylene action (AgNO3) and ethylene synthesis (3,5-diiodo-4-hydroxybenzoic acid and benzyl isothiocyanate; BITC). The production of ethylene from both intact and excised systems was also measured from 0 to 48 h after gravistimulation, relative to vertical controls. Although gravitropic curvature is initiated, and indeed enters the most rapid phase of upward bending during the first 6 h, there is no difference in ethylene production between vertical and geostimulated plants during this period. The ethylene production of gravistimulated plants rises sharply to a maximum at 24 h, then decreases steeply to almost the control level by 48 h, at which time the rate of upward curvature is diminishing. Neither the addition nor removal of ethylene, nor the addition of inhibitors affecting ethylene-action (AgNO3) or synthesis (DIHB) influence gravitropic bending or internodal extension in excised segments. Although the ethylene synthesis inhibitor BITC slowed down the rate of upward bending, this effect could not be reversed by addition of ethylene. We conclude that the burst in ethylene production that develops in leaf-sheath bases (pulvini) after they have started to curve upwards is not primary to the induction of curvature. We further suggest that ethylene has no major effect or role in the induction of upward bending after gravistimulation. The metabolism of high specific activity gibberellin A1 ([3H]-GA1) in the excised system was assessed during 1, 2 and 4 h of gravistimulation. Changes in endogenous GAs and GA metabolism have been shown previously to be correlated (at the later stages) with gravistimulated bending in intact Avena shoots. The excised segments "leaked" free [3H]-GAs and [3H]-GA glucosyl conjugate-like substances into the bathing medium, and this was a confounding factor. Nevertheless, gravistimulated stem segments, and especially the bottom half of the segment, were significantly less leaky then vertical segments. Thus, just 1 h after gravistimulation, bottom segment halves retained 22% more precursor [3H]-GA1, 36% more free [3H]-GA-like metabolites, and 48% more [3H]-GA glucosyl conjugate-like metabolites than vertical segments. In contrast, the 1 h gravistimulated top halves retained slightly less (1-4%) precursor [3H]-GA1 and free [3H]-GA metabolites, but 21% more [3H]-GA glucosyl conjugate-like radioactivity than vertical segments.

Analysis and significance of gravity-induced asymmetric growth in the grass leaf-sheath pulvinus.

The negative gravitropic response in the grass leaf-sheath pulvinus is a consequence of cell elongation involving all cells except those of the uppermost region of the upper flank of an horizontally oriented pulvinus. The lowermost layer of cells elongate maximally, and the regions in between elongate to intermediate extents. The resulting curvatures of a responding pulvinus can be expressed mathematically by relating the angle of curvature (theta) to the original length (L0) and the maximal length of the lower surface (L1) and the diameter of the organ (D), using the equation, theta = (L1-L0)/D, where theta is in radians. The elongation response (S) of any individual cells within the pulvinus can be expressed by the equation, S = 0.5 - r cos theta, where r is the radius of the pulvinus and theta is in degrees. Microscopic measurement of cell lengths in different regions of the pulvinus supports the mathematical predictions. Indirect support is also obtained from the use of colchicine, coumarin, dichlorobenzonitrile (DCBN) and isopropyl N-chlorophenyl carbamate which exaggerate the inherent asymmetry during gravitropic response. Coumarin and DCBN also induce thickenings in the radial walls which appear first in the statenchyma, and later, in cells located towards the outer periphery of the pulvinus. The distribution patterns of these thickenings suggest that the asymmetric growth response of the pulvinus may be due to a differential and radial, centrifugal transport of growth promotors from the central statenchyma region.