Introduction
Many plants orient their leaves in response to directional light signals. Heliotropic
movements, or movements that are affected by the sun, are common among plants belonging
to the families Malvaceae, Fabaceae, Nyctaginaceae, and Oxalidaceae. The leaves of many
plants, including Crotalaria pallida, exhibit diaheliotropic movement. C. pallida is a
woody shrub native to South Africa. Its trifoliate leaves are connected to the petiole
by 3-4 mm long pulvinules (Schmalstig). In diaheliotropic movement, the plant's leaves
are oriented perpendicular to the sun's rays, thereby maximizing the interception of
photosynthetically active radiation (PAR). In some plants, but not all, his response
occurs particularly during the morning and late afternoon, when the light is coming at
more of an angle and the water stress is not as severe (Donahue and Vogelmann). Under
these conditions the lamina of the leaf is within less than 15? from the normal to the
sun. Many plants that exhibit diaheliotropic movements also show paraheliotropic
response as well. Paraheliotropism minimizes water loss by reducing the amount of light
absorbed by the leaves; the leaves orient themselves parallel to the sun's rays. Plants
that exhibit paraheliotropic behavior usually do so at midday, when the sun's rays are
perpendicular to the ground. This reorientation takes place only in leaves of plants that
are capable of nastic light-driven movements, such as the trifoliate leaf of Erythrina
spp. (Herbert 1984). However, this phenomenon has been observed in other legume species
that exhibit diaheliotropic leaf movement as well. Their movement is temporarily
transformed from diaheliotropic to paraheliotropic. In doing so, the interception of
solar radiation is maximized during the morning and late afternoon, and minimized during
midday. The leaves of Crotalaria pallida also exhibit nyctinastic, or sleep, movements,
in which the leaves fold down at night. The solar tracking may also provide a
competitive advantage during early growth, since there is little shading, and also by
intercepting more radiant heat in the early morning, thus raising leaf temperature nearer
the optimum for photosynthesis.
Integral to understanding the heliotropic movements of a plant is determining how the
leaf detects the angle at which the light is incident upon it, how this perception is
transduced to the pulvinus, and finally, how this signal can effect a physiological
response (Donahue and Vogelmann).
In the species Crotalaria pallida, blue light seems to be the wavelength that stimulates
these leaf movements (Scmalstig). It has been implicated in the photonastic unfolding of
leaves and in the diaheliotropic response in Mactroptilium atropurpureum and Lupinus
succulentus (Schwartz, Gilboa, and Koller 1987). However, the light receptor involved
can not be determined from the data. The site of light perception for Crotalaria pallida
is the proximal portion of the lamina. No leaflet movement occurs when the lamina is
shaded and only the pulvinule is exposed to light. However, in many other plant species,
including Phaseolus vulgaris and Glycine max, the site of light perception is the
pulvinule, although these plants are not true suntracking plants. The compound lamina of
Lupinus succulentus does not respond to a directional light signal if its pulvini are
shaded, but it does respond if only the pulvini was exposed. That the pulvinus is the
site for light perception was the accepted theory for many years. However, experiments
with L. palaestinus showed that the proximal 3-4 mm of the lamina needed to be exposed
for a diaheliotropic response to occur. If the light is detected by photoreceptors in
the laminae, somehow this light signal must be transmitted to the cells of the pulvinus.
There are three possible ways this may be done. One is that the light is channeled to
the pulvinus from the lamina. However, this is unlikely since an experiment with oblique
light on the lamina and vertical light on the pulvinus resulted in the lamina responding
to the oblique light. Otherwise, the light coming from the lamina would be drowned out
by the light shining on the pulvinus. Another possibility is that some electrical signal
is transmitted from the lamina to the pulvinus as in Mimosa. It is also possible that
some chemical is transported from the lamina to the pulvinus via the phloem. These
chemicals can be defined as naturally occuring molecules that affect some physiological
process of the plant. They may be active in concentrations as low as 10-5 to 10-7 M
solution. Whatchemical, if any, is used by C. pallida to transmit the light signal from
the lamina of the leaflet to its pulvinule is unknown. Periodic leaf movement factor 1
(PLMF 1) has been isolated from Acacia karroo, a plant with pinnate leaves that exhibits
nychinastic sleep movements, as well as other species of Acacia, Oxalis, and Samanea.
PLNF 1 has also been isolated from Mimosa pudica, as has the molecule M-LMF 5
(Schildknecht).
The movement of the leaflets is effected by the swelling and shrinking of cells on
opposite sides of the pulvinus (Kim, et al.) In nyctinastic plants, cells that take up
water when a leaf rises and lose water when the leaf lowers are called extensor cells.
The opposite occurs in the flexor cells (Satter and Galston). When the extensor cells on
one side of the pulvinus take up water and swell, the flexor cells on the other side
release water and shrink. The opposite of this movement can also occur. However, the
terms extensor and flexor are not rigidly defined. Rather, the regions are defined
according to function, not position. Basically, the pulvini cells that are on the
adaxial (facing the light) side of the pulvinus are the flexor cells, and the cells on
the abaxial side are the extensor cells. Therefore, the terms can mean different cells
in the same pulvinus at varying times of the day. By coordinating these swellings and
shrinkings, the leaves are able to orient themselves perpendicular to the sunlight in
diaheliotropic plants.
Leaf movements are the result of changes in turgor pressure in the pulvinus. The
pulvinus is a small group of cells at the base of the lamina of each leaflet. The
reversible axial expansion and contraction of the extensor and flexor cells take place by
reversible changes in the volume of their motor cells. These result from massive fluxes
of osmotically active solutes across the cell membrane. K+ is the ion that is usually
implicated in this process, and is balanced by the co-transport of Cl- and other organic
and inorganic anions.
While the mechanisms of diaheliotropic leaf movements have not been studied extensively,
much data exists detailing nyctinastic movements. Several ions are believed to be
involved in leaf movment. These include K+, H+, Cl-, malate, and other small organic
anions. K+ is the most abundant ion in pulvini cells. Evidence suggests that
electrogenic ion secretion is responsible for K+ uptake in nyctinastic plants. The
transition from light to darkness activates the H+/ATPase in the flexor cells of the
pulvinus. This leads to the release of bound K+ from the apoplast and movement of the K+
into the cells by way of an ion channel. This increase in K+ in the cell decreases the
osmotic potential of the cells, and water than influxes into the flexor cells, increasing
their volume. In Samanea, K+ levels changed four-fold in flexor cells during the
transition from light to darkness. In a similar experiment, during hour four of a
photoperiod, the extensor apoplast of Samanea had 14mM and the flexor apoplast had 23 mM
of K+. After the lights were turned off, inducing nyctinastic movements, the K+ level in
the apoplast rose to 72 mM in the extensor cells and declined to 10mM in the flexor
cells. Therefore, it appears that swelling cells take up K+ from the apoplast and
shrinking cells release K+ into the apoplast.
In the pulvinus of Samanea saman, depolarization of the plasma membrane opens K+
channels (Kim et al). The driving force for the transport of K+ across the cell
membranes is apparently derived from activity of an electrogenic proton pump. This
creates an electrochemical gradient that allows for K+ movement. From concentration
measurements in pulvini, K+ seems to be the most important ion involved in the volume
changes of these cells. How then, is K+ allowed to be at higher concentrations inside a
cell than out of it? Studies indicate that the K+ channels are not always open. In
protoplasts of Samanea saman, K+ channels were closed when the membrane potential was
below -40mV and open when the membrane potential was depolarized to above -40mV. A
voltage-gated K+ channel that is opened upon depolarization has been observed in every
patch clamp study of the plasma membranes of higher plants, including Samanea motor cells
and Mimosa pulviner cells.
It is proposed that electrogenic H+ secretion results in a proton motive force, a
gradient in pH and in membrane potential, that facilitates the uptake of K+, Cl-,
sucrose, and other anions. External sodium acetate promotes closure and inhibits opening
in Albizzia. This effect could be caused by a decrease in transmembrane pH gradients.
The promotion of opening and inhibition of closure of leaves by fusicoccin and auxin in
Cassia, Mimosa, and Albizzia also implicate H+ in the solute uptake of motor cells, since
both chemicals are H+/ATPase activators, stimulating H+ secretion from the plant cells
into the apoplast. Vanadate, an H+/ATPase inhibitor, inhibits rhythmic leaflet closure
in Albizzia. Although this conflicts with the movement effected by fusicoccin and auxin,
it is believed that vanadate affects different cells, acting upon flexor rather than
extensor cells. The model indicates that there are two possible types of H+ pumps. One
is the electrogenic pump that creates the pmf mentioned above and opens the K+ channels.
The other pump is a H+/K+ exchanger, in which K+ is pumped into the cell as H+ is pumped
out of the cell in a type of antiport. The presence of this typ of pump is only
hypothetical, however, since at present there is no evidence to support it. Thus there
are two possible ways for K+ to enter the pulvini cells. The buildup of the pH gradient
may also promote Cl- entry into the cell via a H+/Cl- cotransporter as the H+ trickles
back into the cell. Cl- ions may also be driven by the electrochemical gradient for Cl-
via Cl- channels, as with K+. A large Cl- channel was observed in the membrane of
Samanea flexor protoplasts. The channel closed at membrane potentials above 50mV and
opened at potentials as low as -100mV.
Light-driven changes in membrane potential may be involved in the activation of these
proton pumps. This may be mediated by effects on cytoplasmic Ca2+. Ca2+-chelators
inhibit the nyctinastic folding as well as the photonastic unfolding responses in Cassia.
Thus Ca2+ may act as a second messenger in a calmodulin-dependent reaction. The Ca2+
may be what turns on the electrogenic proton pumps, causing changes in membrane
potential. However, there is no direct evidence to support this hypothesis, although
chemicals that are known to change calcium levels have been shown to alter the leaf
movement of Cassia fasciculata and other nyctinastic plants. One study involving Samanea
postulates that Ca2+ channels are also present in the plasma membrane of pulvini cells,
and inositol triphoshate, a second messenger in the signal transduction pathway in
animals, stimulates the opening of these channels. This insinuates that some light
signal binds to a receptor on the outside of the cell and stimulates this transduction
pathway. However, whether this hypothesis is true is unclear. It has also been proposed
that an outwardly directed Ca2+ pump functions as a transport mechanism to restore
homeostasis after Ca2+ uptake through channels.
The changes in Cl- levels in the apoplast are less then that for K+. The Cl- levels are
75% that of K+ in Albizzia, 40-80% in Samanea, and 40% in Phaseolus. Therefore, other
negatively charged ions must be used to compensate for the positive charges of the K+.
Malate concentrations vary, and it is lower in shrunken cells than in swollen cells. It
is believed that malate is synthesized when there is not enough Cl- present to counteract
the charges of the K+.
An experiment with soybeans (Cronland) examined the role of K+ channels and H+/ATPase in
the plasma membrane in paraheliotropic movement. This was done by treating the pulvini
with the K+ channel blocker tetraethylammonium chloride (TEA), the H+/ATPase activator
fusicoccin, and the H+/ATPase inhibitors vanadate and erythrosin-B. In all cases the
leaf movements of the plant were inhibited, leading to the hypothesis that the
directional light results in an influx of K+ into the flexor cells from the apoplast and
an efflux of K+ from the extensor cells into the apoplast, and these movements are driven
by H+/ATPase pumps. This combined reaction results in the elevation of the leaflet
towards the light.
In this study, the diheliotropic movements of C. pallida are examined. The purpose of
this experiment is to determine which ions, if any, are used by pulvini cells of
Crotalaria pallida Aiton to control the uptake of water, thereby affecting diheliotropic
movement. As mentioned before, most studies investigating the mechanisms of leaf
movement have been performed on nyctinastic plants. These plants respond to light and
dark changes, not direction or intensity of a light stimulus. Therefore, it is of
interest to learn whether the same principles can be applied to diheliotropic movement.
Different inhibitors at varying concentrations will be injected individually into the
pulvinus of C. pallida, and the suntracking ability of the plant will then be measured.
Tetraethylammonium (TEA), a K+ channel blocker will be added to test whether K+ is
involved in suntracking. Likewise, , a Cl- channel blocker will be added to determine
if Cl- is used. Vanadate, a H+/ATPase inhibitor, will determine if hydrogen ions are
pumped across the plasma membrane, causing a hyperpolarization of the membrane.
Fusicoccin, a H+/ATPase activator will also be tested .
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