Plant
Physiology I: PLS622 2006
Introduction:
Cell
division, expansion, cell-cell communication:
The goals for this lecture include an
understanding of:
1) The basics of
the cell cycle.
2) The rudiments
of control of the cell cycle.
3) The primary
components of the cell wall.
4) The basic
interactions among these components.
5) The
acid-growth theory
6) Apoplastic and
Symplastic information retrieval and propagation.
How
do plants increase in size?
1)
more cells.
2)
larger cells.
How
does plant organ-shape arise?
Organization
of cells creates strict geometric shapes.
The
basics of the cell cycle.
Cell
division:
Arguably one of the most fundamental events of life, cell division depends on a
progenitor increasing its intracellular content, apportioning it, duplicating
its genetic material, dividing it in exact replica, and partitioning one part
of the previous cell from the other, in the process forming two cells with
identical genetic information. This procedure has continued, non-stop, since
the beginning of life, leading to the current model of the cell-division cycle, or cell
cycle (Fig. 1).
Figure
1: The standard cell cycle.
The
mitotic phase of the cell cycle (“M” in the diagram above) is comprised of Prophase, Premetaphase, Metaphase,
Anaphase and Telophase. The
objective of mitosis is to divide the chromatin into two identical chromosome
assemblages and enclose each portion in a nucleus. To do this, the duplicated
chromosomes shorten and thicken (becoming chromatids)
while the nucleolus and nucleus eventually break down signifying the end of
prophase. Next, microtubules (formed
from tubulin polymers) emanate from opposite
poles of the plant cell to form the spindle
(commencement of Metaphase). One important difference between plant and
animal cells is that plant cells do not have centrosomes at the poles to which
the microtubules attach as occurs in animal cells (Fig. 2). The chromatids (duplicated, condensed
chromosomes) attach to the spindles via the kinetochore (centromer).
The chromatids align (or congress) at
the equator of the cell (metaphase plate)
demarcating the end of metaphase. In anaphase, the kinetochores separate and
migrate towards opposite poles, dividing the chromatid into component daughter chromosomes. By the end of
anaphase two identical sets of chromosomes are at opposite poles of the cell.
At this point, the chromosomes de-condense and become once again enclosed in a
nucleus (Telophase). The cell now forms two daughter cells by the formation of
the cell plate (phragmoplast) (Fig. 3)
which can be considered as the termination of telophase.
Figure
2: Animal Mitosis.
Figure
3: Plant Mitosis.
A
specialized form of chromatin apportioning, meiosis, leads to the production of gametes. In the case of a
diploid organism, a single cell replicates its DNA, and then undergoes two
successive divisions which reduces the chromosomal content so that each of the
four resulting cells is haploid (Fig. 4).
The
rudiments of control of the cell cycle.
Considerable research is currently
focussed on elucidating how the events required for successful cell division
are regulated. The G-phases of the
cell cycle have been determined to be periods of rest for the cell, providing
check points determining whether the next phase of the cycle is entered and, if
so, when. There is at least one major check in G1 and G2,
both of which cue on the size of the cell and, in yeast, the environmental
conditions conducive to cellular proliferation. Central to this control are the
cyclin-dependent protein kinases (cdks)
that can phosphorylate proteins (thereby changing their activation) responsible
for forcing cells into the next phase of the cell cycle. The cdk’s are present
at all stages of the cell cycle but, as their name implies, are dependent on
the presence of a second family of labile proteins called cyclins for their phosphorylating ability. There are two types of
cyclins, mitotic cyclins that
control entry into the mitotic phase
of the cell cycle and G1
cyclins that control exit from G1 and entry into the S-phase. These proteins undergo rapid
accumulation when conditions are favorable for entry into the mitotic- and
S-phases of the cell cycle, respectively, and are rapidly degraded once the
cell has committed to entry into the respective phase.
The root meristemless mutants
of arabidopsis (rml1 and rml2)
provided a glimpse into just how complex the control of cell division in plants
can get. These mutants result in primary roots of less than 2 mm length due to
a lack of cell division in the root apical meristem following germination.
However, the shoot is capable of normal cell division as is callus. It was
found that the rml1 defect was in a
gene in the glutathione biosynthetic pathway. Further investigation into the
lack of cell division in rml1 plants
defined an absolute requirement for adequate levels of glutathione for the G1-to-S
phase transition. Apparently, glutathione is not necessary for cell division in
the shoot apical meristem or it is synthesized by another, non-mutant gene.
Figure
4: Meiosis.
How do you stop dividing?:
- In vertebrate cells, withdrawal from
the cell cycle is mediated by cdk’s. The degradation of mitotic cyclin and the
absence of G1 cyclin traps the newly formed daughter cells in the G1
resting phase. In plants cells there
is evidence to suggest that the same mechanism may be in place. Cyclin-dependent kinase inhibitors
(CDIs) have been identified that result in maize endosperm arrest.
Formative vs proliferative cell
division.
-
Formative cell
division
serves to create cells that will eventually take on distinctly different fates.
-
Proliferative
division
serves to increase the number of the same type of cell.
The
primary components of the cell wall.
Cell expansion:
Water
uptake drives cell expansion in plants which is an irreversible increase in
cell volume. By taking up water into the central vacuole, plants have developed
an economical method of increasing their size by orders of magnitude while
maintaining approximately the same cytoplasmic volume, albeit, now dispersed
over the periphery of a larger cell. Water uptake into the vacuole to increase
turgor will not be effective in driving cell expansion unless the rigid cell
wall is somehow induced to weaken, allowing the pressure within to force the
cell wall to extend. This is not analogous to air forcing a balloon to expand
since the wall of the balloon becomes increasingly thin until it ultimately
fails. The plant cell wall deposits and incorporates new wall material into the
expanding cell wall so that no "thinning" occurs. The ability of the
cell wall to expand and incorporate new cell wall material while under stress
is the result of its complex makeup and precise partial disassembly by some
select wall modifying enzymes.
The plant cell wall is thought to be
comprised of two major classes of components, the cellulose-hemicellulose
network and the pectic network. The former is cemented within the matrix of the
latter.
Figure 5
+ 6: Cellulose synthesis at the plasmamembrane.
There
are five main components to plant cell walls which in dicots have the following
approximate percent compositions; 1) cellulose (~30% wall dry weight); 2)
hemicelluloses (~30% wall dry weight); 3) pectins (~35% wall dry weight); 4)
proteins (~1-5% wall dry weight) and; 5) water (~75% wall wet weight). Ions
such as calcium make up a sixth component but in truly negligible amounts
relative to the first 5.
The
basic interactions among these components.
Cellulose is the
backbone of the cell wall, imparting, depending on the predominant orientation
of the fibrils, the wall’s ability to undergo expansion or not. In those
instances where cellulose microfibril orientation predisposes the wall to
expansion, fibril orientation also dictates the directionality of this
expansion. Cellulose in the cell wall is a rigid semi-crystalline polymer that
is laid down on the inner cell wall surface (outer surface of the
plasmamembrane) by a complex assemblage of proteins arranged in a rosette often
referred to as the terminal rosette
(Fig. 5). The rosette is embedded in the plasmamembrane, spanning it and
presumably anchored to and travelling along tubulin microtubules aligned on the
inner surface of the cell membrane (Fig. 6). How the cellulose microfibrils are
arranged determines if and how the cell expands.
Figure 7: Diagram of
the plant cell wall from two different cells, one that is not expanding or
expanding isotropically and a second expanding directionally or
anisotropically.
If
the microfibrils are secreted in a non-parallel manner (isotropically), then
the microfibrils may prevent cell expansion since there is no direction the
cell may extend without breaking a microfibril (Fig. 7). To date, there have
been no reports of cellulases produced by plants that are capable of
hydrolyzing crystalline microfibrils. The cellulases that have been discovered
are all thought to act on hemicellulose substrates such as xyloglucan. The
single exception is a putatively membrane-bound cellulase involved in cellulose
synthesis, presumably by hydrolysing a single 1-4 glucan chain from the
cellulose synthesizing apparatus comprising one component of a rosette (Fig. 5
and 6). Alternatively, in response to internal signals, microtubules can
arrange themselves in parallel with the result that the enzyme rosettes
extruding cellulose microfibrils to the inner wall surface exterior to the
plasmamembrane lay down the microfibrils in parallel (anisotropically, Fig. 7). Hence, the configuration of the cellulose
in the stress bearing region of the cell wall resembles a spring or slinky. It
is difficult to expand the spring laterally but much easier to extend the
spring longitudinally. Recently, a mutation in the membrane-bound cellulase
involved in cellulose synthesis has been isolated. Dubbed korrigan (kor),
the mutant plants are dwarfed and exhibit aberrant cell expansion.
The
hemicellulosic component of plant
cell walls is thought to be intimately associated with the cellulose
microfibrils, either laying along the microfibrils for much of their length
associated by hydrogen bonds, or actually embedded in the interior of the
microfibrils. The hemicellulosic constituents are delivered to the cell wall
from Golgi derived vesicles in which they are synthesised. Upon arrival at the
plasma membrane the vesicles fuse with the membrane and release the contents
into the wall. These wall components are then somehow incorporated into the
stress bearing wall, probably being linked together enzymatically to form
longer, more highly branched polymers than were synthesized in the Golgi. This
process appears to be operational in the deposition of xyloglucan into the cell
wall of plants using xyloglucan
endo-transglycosylase (XET) to
polymerize short, vesicle-deposited xyloglucan chains into larger polymers of
xyloglucan. Additionally, the simultaneous extrusion of semi-crystalline
cellulose at the inner wall surface and a marked affinity of xyloglucan for
cellulose ensures their tight association, even the possibility of xyloglucan
embedding itself into the elongating microfibril. To permit cell wall
expansion, these hemicelluloses must be either disassociated from the cellulose
microfibrils and themselves by a recently discovered enzyme christened “expansin” or disentangled from the
matrix usually though partial polymer hydrolysis by endo-glycosylases (EGases).
Pectin forms a “gel”
in which the other cell wall components are embedded. Although it is most
abundant in the middle lamella
between cells, cementing them together, pectin is found throughout the plant
cell wall. Pectin is initially deposited at the inner cell wall surface in
discrete lengths by Golgi derived vesicles where it is formed. This pectin is
highly methyl esterified when
deposited making it resistant to hydrolysis and relatively unreactive with cell
wall calcium and other ions. Upon deposition, the pectin is typically
de-esterified by pectin methyl esterase
(PME) imparting to it a negative
charge and allowing it to form rigid configurations and bind tenaciously to
calcium. It is also now susceptible to modification by pectin hydrolysing
enzymes (polygalacturonases, PGs).
The degree to which pectin is hydrated, its degree of esterification, and the
bonds it is participating in are all thought to control wall porosity which in
turn determines the sieve size of the cell wall, dictating what size particle,
protein or polysaccharide, can pass through the matrix.
Protein
in the cell wall can be structural, such as extensins thought to impart inflexibility to the cell wall, or
enzymatic, such as expansins, XETs, EGases, PMEs, PGs, etc. Although they
comprise only a small percentage of the total cell wall by dry weight, they are
absolutely crucial to its ability to expand.
Water is often
ignored as a major component of the living, dynamic plant cell wall but,
besides imparting solvent and lubrication, it also determines the concentration
of the components of the wall and their hydration determines wall porosity. Its
influx into the vacuole of cells dictates the turgor they are capable of
exerting and no plant cell, however disposed to cell wall loosening, can expand
without turgor pressure.
Plant cell
walls isolated from regions undergoing natural expansion are susceptible to
elongation due to a decrease in pH, so-called acid growth (see assigned
reading; Hager 2003. J. Plant Res. 116: 483-505 parts thereof). Acid growth is
thought to to be induced by the plant hormone auxin by the stimulation of
plasma membrane H+-ATPases that pump protons from the cytoplasm out
of the cell thereby acidifying the apoplast. This stimulates the cell wall
modifying enzymes allowing wall components to "creep" past each other
and growth to occur. Additionally, inward rectifying potassium channels are
activated to allow potassium ions into the cell to balance the charge
differential across the plasma membrane resulting from H+ efflux.
This eventually results in a decrease in vacuolar osmotic potential, the influx
of water, and maintenance of turgor in the expanding cell. Protease treatment
has been shown to eliminate acid growth from plant cell walls normally
responding to decreased pH. This led to Daniel Cosgrove isolating two proteins
from cucumber hypocotyls that potentiate acid
growth. These were named expansins
and are purportedly solely responsible for acid growth of plant cell walls
without cleaving any cell wall constituent. The family of expansin genes in
plants is large with different members putatively involved in a diversity of
processes that require cell expansion. In Arabidopsis, alterations in the
expression of one expansin family member, AtEXP10,
results in aberrant leaf morphology and pedicel abscission. The
expansin-mediated growth of plant cell walls is enhanced by pretreatment of
cell walls by hemicellulases and presumably this is also the case in vivo,
but the hemicellulases tested to date contribute little to cell wall expansion
relative to expansin. Yet, in plant parts that normally do not expand, the
cells are resistant to acid-mediated elongation. There is therefore, a point in
the life of a cell where it becomes rigidly fixed in size and unresponsive to
molecules and environments that promoted cell elongation in the tissue at some
earlier developmental stage. This is termed rigidifying the cell wall and
generally fixes cell size irreversibly. The process of regidification is
thought to involve the reduction of cell wall loosening processes, a more
comprehensive cross-linking of cell wall components resisting cell wall
expansion, and an alteration in the components of the cell wall, stiffening it
against extension.
Cell-cell communication:
Apoplastic
and Symplastic information retrieval and propagation.
Communication
among cells takes place both apoplastically and symplastically in the plant (apoplast and symplast defined on the required terms page). Apoplastic signaling
is dependent on the assembly and accessibility of a variety of receptors on the
plasmalemma (plasma membrane). The receptor must span the plasma membrane
completely and be capable of initiating a biochemical change on the interior
surface as a consequence of stimulation on the exterior surface, usually by
interaction with, and/or attachment to other proteins.
The
former event is depicted by the ethylene receptors. These receptors have an
N-terminus that contains three membrane-spanning domains that embed the
receptor in the membrane and also serve to bind ethylene in the presence of a
copper cofactor. These proteins span the plasma membrane as homodimers. The
presence of ethylene at the surface of the cell is communicated to the interior
of the cell by a change in the phosphorylation status of the receptors
cytoplasmic surface. This, in turn, triggers further signal transduction,
ultimately changing gene expression in the nucleus.
The
second form of receptor is best depicted by a receptor that, when stimulated,
activates phospholipase-C. This is a very common form of receptor in animal
cells. The activated phospholipase-C hydrolyses inositol containing
phospho-lipids, liberating a number of bio-active "secondary
messengers" on the cytoplasmic face of the membrane which stimulate a
signal cascade that results in a change in gene expression.
Cell-cell
communication in plants includes the regulation of plant growth and development
by phytohormones, the receptor-ligand signaling evident in pollen-stigma
interactions leading to self-incompatibility responses, and intercellular
trafficking of substances through plasmodesmata (PD). PD are unique to the
plant, a consequence of how cell division occurs in these organisms. The fact
that: 1) there is incomplete separation of the cytoplasm during cytokinesis
and; 2) a cell plate forms de novo
across the cell but leaves some regions of cytoplasmic continuity between the
two daughter cells, gives rise to plasmodesmata between the cell clones. Hence,
every set of daughter cells are symplastically continuous through what has
become known as primary, unbranched PD.
Cells that are not derived from the same mother cell can also form PD
connections and do so frequently. The mechanism by which these PD are formed
varies from that of primary PD in that secondary
branched PD can form de novo in
preexisting cell walls. They usually consist of multiple cytoplasmic strands
that converge between the two cells in a central cavity before branching into
multiple channels again to enter the second cell.
Symplastic
domains:
The cells of the embryos of those plants studied to date have been shown to be
all symplastically continuous. However, soon after the completion of
germination, this symplastic continuity is disrupted so that some assemblages
of cells in the seedling lose their PD connections with other assemblages. This
establishes groups of cells that, although in direct symplastic contact with
each other, are isolated symplastically from additional groups of cells setting
up domains of cells that, although in close proximity to each other may be in
very different physiological states. The symplastic continuity of cells and
their isolation from neighboring cells has vast implications for plant cell
differentiation which is dependent on cell position. For example, the guard
cells comprising the stomata are completely symplastically isolated by the time
the stomata are completely differentiated. The PD connections between the guard
cells and their surrounding cells are lost though protein degradation of PD components.
In addition, evidence from cell ablation studies has shown that more mature
cells in the plant root tip dictate to recently divided cells more distal in
the same file, their developmental fate. Moreover, there exists a mechanism to
dictate the direction of propagation of such a signal so that laterally
adjacent cells are unaffected by signals produced in the more mature cell, only
those cells within the same file and less mature than the cell producing the
signal are influenced. One mechanism allowing this type of developmental
control would be to have the cells within a file, all clones of their initial
at the root apex, symplastically continuous with each other through primary PD
and symplastically isolated from adjacent files of cells. This is only a
hypothesis but is a possible explanation for this developmental phenomenon. In
support of this hypothesis are two observations made with diffusable dyes. The
first is that the more mature cells of the arabidopsis epidermis are
symplastically isolated from the underlying cells of the cortex. Additionally,
root hairs are also symplastically isolated from the epidermal cells
surrounding them. A mutant in cell wall development (knolle) artificially
maintains symplastic connections between symplastic domains not normally
associated. As a probable result, the knolle
mutant has many abnormalities in development.
Plasmodesmatal
connections can also undergo developmental changes during the differentiation
of plant cells. In young buds and flowers of Setcreasea purpurea the PD size exclusion limits (SEL) of stamen
hairs are less than 1000
The
developmental changes that regulate PD function and SELs has profound
implications for plant-virus interactions. Plant viruses have been shown to be
capable of altering the SEL of PD considerably. However, this ability to alter
SEL is modified by host developmental stage and physiological state. Hence,
arabidopsis plants grown under long-days mature early and are resistant to the
cauliflower mosaic virus (CaMV) while those grown under short-days mature
slowly and are not as resistant to CaMV infection. By anology, if pathogenic
molecules/organisms can move systemically though PD, then it is anticipated
that endogenous protective molecules, so called pathogen related (PR)
molecules, may also turn out to be targeted to PD for further transport into
neighboring cells.
Figure 8: Diagramatic representation of one model of
plasmodesmata structure.
Plasmodesmata
are complex organelles, certainly not a simple "hole-in-the-wall".
The cytoplasmic sleeve of plasmodesmata appears to be subdivided into many
smaller microchannels (Fig. 8). The "spokes" detected in the lumen of
the plasmodesmata are probably comprised of actin, a protein that has been
localized to the PD. Alterations in the size exclusion limit of PD has led to
intensive study of various viral and plant proteins thought to function in the
modification of the SEL of the PD. These proteins are capable of transporting
select, very large proteins and nucleic acid polymers through the
plasmodesmata. These so called movement proteins are currently under intense
scrutiny to determine how, mechanistically, they permit such trafficking. With
their discovery has come the realization that the PD probably play a hither to
vastly undervalued role in cell-to-cell communication in plants.
Models of transport through PD: Cell
biologists use inert, usually fluorescent, dextrans of precisely defined size
to determine the SEL of plasmodesmata. Using these dextrans in concert with
plant or viral movement proteins has revealed some interesting results
regarding the probably method of transport through PD. Evidence exists that
there is more than one type of protein facilitated movement through PD.
Relatively short distances between mesophyll cells leads to short PD which,
upon being dilated by movement proteins in the presence of large dextran
molecules, permit the transport of both since the whole route is gated open
simultaneously. However, longer PD linking trichome cells with other cells may
either; 1) be capable of unfolding proteins with associated transport signals
and transporting them through the undilated PD to refold on the other side or;
2) gate the PD open very quickly and propagate the open state of the PD along
the PD so that the PD is not open from neck to neck simultaneously in response
to a movement protein signal. Either scenario results in the trafficking of the
movement protein only and the exclusion of the dextrans from transport into
neighboring cells unlike movement through PD in mesophyll cells.
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