Untitled Essay, Research Paper
Involvement of K+ in Leaf Movements During SuntrackingIntroduction
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
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 .