All posts by Chris Wolverton

Organ Formation

Although the most primitive land plants possessed little more than stem-like structures, most land plants today produce broad, flat leaves that maximize the surface area exposed to light. While it may be clear how meristem-driven growth can result in the formation of an axial organ, like a stem or root, it is probably harder to imagine how a meristem could produce a laminar structure like a leaf.

The first visible indication that a leaf is about to form is a tiny bump on the surface of the apical dome, the tip-most section of the elongating stem. These bumps form off to one side of the dome, and the position of the bump determines the pattern of leaf placement along the stem, called phyllotaxy. In other words, for a species having alternate phyllotaxy, the position of consecutive bumps would spiral around the apical dome, while species with opposite phyllotaxy would form two bumps simultaneously and offset by 180° from each other. As time passes, the bumps, known as leaf primordia, expand into leaves, and the leaves become separated by expansion of the stem over the time between which the bumps formed.

Given that the arrangement of leaves on a stem is highly reproducible within a species, the placement of each leaf primordium must be regulated. Over the last 20 years, there has been substantial progress made in identifying some of the factors that control maintenance of the meristematic population as well as initiation of new leaf primordia. One of the key concepts to emerge is that, while the meristem looks like an amorphous mass of cells, it is actually organized into two zones based on the expression patterns of particular classes of genes, many of which encode transcription factors. These two zones, known as the central zone (CZ) and peripheral zone (PZ), exist in a dynamic tension, such that they tend to protect their cellular ‘turf’ by preventing expression of genes from the other zone. In addition to zones, the shoot apical meristem is also partitioned into layers, with most species having 3 layers (L1, L2, and L3).

Leaf primordia form in the peripheral zone, and even before a bump is visible, the region where a primordium will form shows downregulation of a transcription factor associated with meristem activity, the STM gene in Arabidopsis. Shortly thereafter, this region begins to protrude from the surface as a leaf primordium. The L1 layer, which is the outermost layer of cells, will form the dermal tissue of the new leaf, while the L3 layer will form the vascular tissues. L2 goes on to differentiate into the rest of the tissues that make up the leaf, most of which are ground tissue. As the leaf primordium is beginning to emerge, cell divisions in L1 and L2 occur perpendicular to the surface of the apex, while those in L3 occur in both perpendicular and parallel planes.

In addition to an important role for transcription factors and signal peptides, plant hormones also have prominent roles in the emergence of leaf primordia. Using an auxin-responsive reporter gene, it has been determined that leaf primordia are local sites of auxin maxima. Auxin, or indole-acetic acid (IAA) is synthesized in existing primordia and transported across the apical dome, where it appears to accumulate and coincide with the site of primordium formation. This accumulation requires a system of polar auxin transporters, which have now been shown to direct auxin transport across the shoot apex to the site of incipient primordium formation. As the new primordium emerges, it exports the auxin necessary to form a new maximum on the other side of the meristem, where a new auxin maximum will form. In other words, as primordia mature, they become auxin sources rather than auxin sinks.

Barton MK (2010) Twenty years on: The inner workings of the shoot apical meristem, a developmental dynamo. Developmental Biology 341:95-113

Water Potential

For cell expansion to continue, cells must continuously take up water from their surroundings. The movement of water occurs in response to several forces, including its concentration, pressure, position in the gravity field, degree of association with soil particles, etc.

Kinds of water flow

The driving force for much of water’s movement at the cellular level is diffusion, the random intermingling of molecules as they collide with like molecules and change directions. Because of these collisions, over time, diffusion tends to result in the movement of molecules from an area of higher concentration to an area of lower concentration. The force behind such a directional movement is therefore the concentration gradient of the molecule. While we are accustomed to thinking about the concentration of a solute, such as a salt, dissolved in a solvent, it is also just as appropriate to speak in terms of the concentration of the solvent itself, as we will see.

Diffusion is a very effective means of movement, but only across short distances or within small volumes. For example, a small molecule in aqueous solution can diffuse 50 μm in about 0.6 s. However, for that same molecule to diffuse 1 m would require 2.5 x 108 seconds, or about 8 yrs! Thankfully, plants are not limited to diffusion as a means of water transport, but also employ bulk flow, which is the movement of a mass of molecules, often in response to a pressure gradient. This kind of transport is employed by the vascular tissues of the plant to accomplish transport across great distances.

A special kind of diffusion called osmosis occurs in living systems, when a selectively permeable membrane is involved. Very often, forces besides solvent concentration influence osmotic flow, with the sum of all such forces represented by the free energy of the solvent. If diffusion is in response to a concentration gradient, and bulk flow a pressure gradient, osmosis is in response to the sum of both of these forces and other influences, all of which together describe water potential.

Water Potential

Water potential is a term denoting the chemical potential of water, which is to say the free energy of water — the energy available to do work. This energy is expressed in units of energy per unit volume J m-3, which is equivalent to pressure (in MPa). There are many contributing factors to water potential, as we’ve seen above. Always keep in mind that pure water has a water potential of 0 (\Psi_w = 0)

The solute potential (\Psi_s) represents the effect of any solutes on the free energy of water. The addition of a solute to water decreases the concentration of water, thus reducing its free energy. The degree of this reduction is a function of the final concentration of solute particles:

\Psi _s = -cRT

where c is in units of osmolality (moles of total dissolved solutes per L). Note the negative sign, a reminder that solutes lower water potential.

The pressure potential (\Psi_p) represents the effect of pressure on the free energy of water; positive pressure increases free energy, and negative pressure reduces it. Pressure within a typical leaf or root cell is positive, while that within the xylem is negative. A cell with \Psi_p = 0 is flaccid.

For some tall plants, gravity becomes an important consideration in predicting total water movement, thus it is necessary to consider the gravity potential (\Psi_g) in such a plant. Each 10 m in vertical distance translates to a 0.1 MPa change in water potential.

Under some conditions, the matric potential (\Psi_m) plays a significant role in determining the flow of water. When water is bound to a surface in a very thin layer, that water is less available to do work, reducing its free energy. This parameter plays an important role during seed germination and under drought conditions.

The effect of all of these factors on the overall water potential is additive, and can be expressed as follows:

\Delta \Psi_w = \Delta \Psi_s + \Delta \Psi_p + \Delta \Psi_g + \Delta \Psi_m

Under most circumstances, the first two terms, corresponding to solute and pressure potentials, are sufficient to determine plant water potential. Plant water potential governs transport of water across membranes, with water always moving from an area of high to low water potential.

Cell expansion

It should be clear by now that the addition of new cells by division in meristematic zones represents an important component of growth. But the dividing of a mother cell in two does not necessarily result in an increase in size. For such an increase to occur, division must be coupled with cell expansion. Without expansion, a dividing cell is just partitioned into smaller and smaller elements.

The force that powers cell expansion is turgor pressure, a positive hydrostatic pressure that builds up within the cell due to the uptake of water. A typical leaf cell in a well-watered plant has a turgor pressure between 0.1 and 3.0 MPa (MPa = megapascals). For reference, a typical car tire is inflated to 30 psi (pounds per square inch), which is equivalent to 0.2 MPa. While such a tremendous pressure would cause most cells to burst, plant cells maintain such high pressures through the resistance of the cell wall.

Cell expansion, then, represents a carefully-controlled balance between wall resistance and wall yielding. For each cell, there is a minimum of pressure required to allow expansion, and above that minimum, expansion proceeds depending on the wall extensibility:

\frac{dV}{dt} = m (P - Y)

where the rate of change in volume, \frac{dV}{dt}, is equal to the product of effective pressure (turgor pressure (P) minus wall yield threshold (Y)) and wall extensibility (m). This is a version of the Lockhart equation, first published in 19651.

Of these factors, the cell has direct temporal control over wall extensibility. One means of regulation over extensibility is by acidification of the cell wall through proton pumping. Lowering the pH of the wall is associated with increased extension. Another means of control is through the production, secretion, and stimulation of wall-bound proteins that loosen bonds between the structural components of the wall. Foremost among these is a family of proteins called expansins, that appear to temporarily loosen the non-covalent linkages between cellulose and other wall polysaccharides. By loosening these linkages, expansins allow the load-bearing members of the wall to slide past each other and permit cell expansion in a very controlled manner.

The wall loosening carried out by the expansins takes place in specific wall locations under specific conditions, allowing the cell to control the timing of expansion and ultimate shape of the cell. Without such control, cells would be free to expand in any direction and at any time, which would have clear implications for tissue and organ form in the mature plant. In addition, the direction of cell expansion is heavily influenced by the pattern of deposition of cellulose microfibrils, which can also serve to constrain the kinds of final shapes a particular cell can achieve.

There is a significant brake in this system of turgor-driven cell expansion, and that is a constraint imposed by biophysics. As turgor pressure drives cell expansion, the volume of the cell increases. This increase in volume reduces the turgor pressure pushing on the wall and providing the force for expansion. Because even a slight increase in volume reduces turgor pressure, water must be taken up continuously to maintain the turgor pressure required to sustain growth.

  1. Lockhart, J.A. (1965) An analysis of irreversible plant cell elongation. Journal of Theoretical Biology 8:264-275. 

Cell fate determination

The question of what the derivative cell will become after it leaves the meristem is governed by its position within the organ. The question of cell fate has been of interest to plant scientists for centuries, but there was little way to test the competing hypotheses of cell lineage and cell position. In an elegant series of experiments, researchers have shown that plant cells can change their fate by being forced into a new position, taking their cues for which kind of cell to form based on their neighbors rather than the identity of their mother cell. Since that set of experiments, researchers have started to identify the signals that transmit this identity information between adjacent cells, largely through microscopic analysis of mutants.

At the broad end of the spectrum, all plant cells fall into one of three tissues with respect to identity: vascular, dermal, and ground tissues. Vascular tissue is composed of cells involved in transport and cells that support transport. Dermal tissue is composed of cells on the surface of the plant. Ground tissue is composed of cells that make up the rest of the plant – in the interior of leaves, stems, and roots but not having a specific role in transport. Thus, the fate of any particular derivative to become a cell in either of these three tissue systems is determined by the location the derivative finds itself within the meristem.

Because of the centrality of position in determining cell fate, one of the most important facets of plant development is the specification of the plane of cell division. For any given mother cell undergoing division, the orientation of the plane of division could mean the difference between a derivative becoming an epidermal cell (dermal tissue) or a cortex cell (ground tissue). This is the case not only because plant cells don’t migrate and adhere, and not only because of the primary role of position in determining cell fate, but also because of the presence of the thick, rigid, cellulosic cell wall. Once wall deposition begins during cytokinesis, there is no chance for a cell to change its position, and hence its fate.

Within the meristem, cells pass through the cell cycle with relatively high frequency. While the process of mitosis has some notable differences from other eukaryotic cells, cytokinesis in plant cells is almost wholly unlike that seen in other eukaryotes. The “textbook” cell undergoes cytokinesis through the familiar ‘pinching in’ of the plasma membrane, but the rigid wall of plant cells is not conducive to such a process. Rather, cytokinesis progresses by the deposition of polysaccharides and membrane lipids at the site of the new cell wall through vesicle trafficking. These vesicles travel along a specialized arrangement of microtubules known as the phragmoplast. Imagine dividing a room in two by suspending a piece of plaster in mid-air, then adding to it until you had built a complete wall, and you would have some idea of how cytokinesis in plants occurs.


Although the plant emerges from embryogenesis with few outwardly visible structures, there is nonetheless already a significant amount of organization in place in the plant embryo. Chief among the organizing structures are two apical meristems, one at the shoot tip and another at the root tip. From these two regions will come all future branches, roots, leaves, and flowers.

light micrograph of the outline of root cells
Root tip of Arabidopsis thaliana, showing the organization of cell files in distinct lineages from the meristem population.

Apical meristems are collections of dividing, undifferentiated cells. When a meristematic cell divides into two daughter cells, one of them is known as an initial, and the other is known as a derivative. Assuming the meristem is neither increasing or decreasing in size over time, only one of these two daughter cells – the initial – will remain in the meristem. The derivative cell will “leave” the meristem eventually, not by cell migration as seen in animals, but by being pushed farther and farther from the meristem proper by continued rounds of division and expansion. As you study the image of a root apex at right, notice how cells form files that almost seem to ‘flow’ from the tip toward the base.

Meristems are therefore a source of new, undifferentiated cells in the tips of growing stems and roots. As they are pushed out of the meristem through division and expansion, these ‘blank slate’ cells begin to take on a cell identity as one of the three major tissue types: dermal, ground, or vascular. In addition to adopting a cell identity, each cell takes on a shape appropriate for its type and location. The shape of individual cells provides the driving force for the formation of organs like leaves or petals.

Principles of Growth

Plants follow a fundamentally different process for growth than animals. For example, it would be odd (to say the least) to find a new limb forming on a person after embryonic development, yet plants produce nearly all of their form post-embryonically, with only a minimal set of body parts created during embryogenesis. The ability to continue producing organs after embryo formation demonstrates that much of plant growth is indeterminate, meaning a plant can continue producing new organs like leaves over and over. This strategy has proven to be flexible and adaptable across a wide range of ecosystems, allowing some plants to live for hundreds, or even thousands, of years.

Ginkgo embryo and gametophyte

In addition to indeterminate growth, plants also show a high degree of developmental plasticity. For an organism that is not free to run away from drought, cold, or herbivores, this attribute is an important strategy. No two plants ever look exactly the same because of this plasticity, and this property allows individuals to become tailor-made for their specific site, producing roots in just the right locations, avoiding rocks and other roots, and sending out branches with leaves right where they will intercept light.

There are, however, certain patterns that seem to be repeated when one looks closely at a plant. The spacing of leaves along a stem, for example, tends to follow a fairly repeatable pattern. This and other patterns indicate that development in plants is iterative, suggesting a pre-recorded program that repeats itself after a certain period.

Plant growth follows these three principles – it is indeterminate, plastic, and iterative. The application of these principles leads to the tremendous variety of forms we find in the plant kingdom, from the arrangement of petals in a flower to the spiral alignment of branches on a pine tree. And all of these arise from the organization of cells into form-producing factories known as meristems.



Phytochrome is known as a photoreversible photoreceptor, meaning that it can change forms from the inactive to active states (and vice-versa) by absorbing distinct wavelengths of light. When it absorbs red light, the phytochrome complex changes shape and becomes activated. At the same time, this change in shape means that it no longer absorbs in the red part of the spectrum, but its absorbance maximum has shifted to the far-red part of the spectrum. Interception of a photon in the far-red energy range will cause the complex to change shape back to the red-absorbing form, as will a prolonged period in the dark.

When embryo-dormant seeds with thin seed coats are exposed to red light in the environment, they are induced to begin germinating through the activation of phytochrome. And because phytochrome is photo-reversible, germination can be halted if the embryo detects that it is in the presence of lower energy far-red light. This watershed observation about germination was one of the first indications of the existence of the phytochrome pathway during the 1950’s and 1960’s:

Seed hit with red light germinated unless it was then hit with far-red; but if red again ensued, it would germinate. Incredibly, all that mattered was which color came last even if the seed was struck by 100 alternating cycles of red and far-red.

Phytochrome structure

Functional phytochrome molecules are composed of a protein component with a pigment molecule bound tightly to it. The apoprotein, as it is known, is encoded by one of five genes in Arabidopsis, and the chromophore, as the pigment is known, is a bilin-type pigment similar in structure to a chlorophyll precursor. Furthermore, phytochromes pair up with another phytochrome to form a dimer. This interaction happens between amino acids at the amino-terminal end of the protein. The other end of the protein, the carboxy-terminal end, shows homology to bacterial histidine kinase domains, but it isn’t clear whether phytochromes act as kinases themselves when activated.

Phytochrome signaling

Upon perception of red light, phytochrome molecules undergo a conformational change and appear to traffic to the nucleus, where they can interact with partner proteins. One such partner is PIF3 (Phytochrome Interacting Factor 3), a basic helix-loop-helix transcription factor that binds to the promoter of light-regulated genes. At least several of the genes regulated by PIF3 encode transcription factors themselves. In addition, phytochrome has been shown to increase expression of at least one gene encoding an enzyme in the GA biosynthetic pathway. GA biosynthesis, in turn, can promote the expression of hydrolytic enzymes associated with energy mobilization and uptake by the embryo.


Toyomasu et al. (1998) Phytochrome Regulates Gibberellin Biosynthesis during Germination of Photoblastic Lettuce Seeds. Plant Physiol 118: 1517-1523

Hormone Perception


As previously discussed, high levels of ABA promote embryo dormancy, while a reduction in ABA concentration leads to an increase in GA biosynthesis and the promotion of germination. But how does the concentration of small molecules make a difference in the cell? These molecules have specific cellular receptors, capable of tightly binding the hormone and activating a signal transduction cascade. The end results of the cascade differ depending on the hormone, but almost always lead to a change in gene expression.

ABA perception

ABA perception facilitates the inactivation of a phosphatase inhibitor.

At least one ABA receptor has been identified to date in plants. The PYR1 gene encodes a protein that can bind ABA and inactivate a protein phosphatase in the PP2C class. Protein phosphatases remove phosphate groups from target proteins, often inactivating them. The result of inactivating this phosphatase is to stimulate ABA responses, the earliest of which include activation of ABA-specific transcription factors. So we can think of ABA as inhibiting an inhibitor of ABA signaling — when the inhibitor (a PP2C protein) is inhibited, ABA signaling can proceed.

A receptor for ABA has been sought for many years, but had eluded researchers until recently. The PYR1 gene was isolated in a screen for mutants that showed resistance to a synthetic compound called pyrabactin, an agonist of ABA responses. This kind of approach, called chemical genetics, is one way to side-step the functional redundancy shown by many plant genes due to genome duplication, and is described in more detail by the Cutler Lab, which successfully employed this approach to uncover PYR1.

GA perception

Recent evidence suggests that a protein called GID1 acts as a GA receptor. When GA binds to GID1, it causes the targeted degradation of a group of proteins known as the DELLA family that normally represses GA responses. By getting DELLA proteins out of the way, the cell can begin to mount a response to the presence of GA. This system of targeted degradation uses a pathway known as the 26S proteasome, a large, multi-subunit cellular garbage disposal. To guard against accidental recycling of proteins, the garbage disposal only accepts proteins with a special tag attached. The tag is itself a small protein called ubiquitin, and the closely regulated process of adding the tag to a target protein is called ubiquitination. The GA receptor GID1 is itself a part of the ubiquitinating machinery and acts by causing the ubiquitination of DELLA proteins. To help visualize this process, I have created an animation showing the interaction of these participants in GA signaling.

Responses to these 2 hormones during dormancy and germination are representative of 2 kinds of signal transduction pathways — kinase cascades and ubiquitin-mediated proteolysis. Both kinds of pathways play crucial roles across the domains of life in mediating responses to signals, and there are many more examples of each within the plant kingdom.


  1. ABA receptor…this time for real?

  2. S. Y. Park, P. Fung, N. Nishimura, D. R. Jensen, H. Fujii et al. (2009) Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324, 1068-1071.

  3. Y. Ma, I. Szostkiewicz, A. Korte, D. Moes, Y. Yang et al. (2009) Regulators of PP2C phosphatase activity function as abscisic acid sensors. 
Science 324, 1064-1068.

  4. Ueguchi-Tanaka, M. et al. (2005) GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin. Nature 437, 693–698

Dormancy & Germination


After completing embryogenesis and seed maturation, most seeds enter into a state of dormancy, which can be thought of as something like “suspended animation”. Dormancy is characterized by extremely slow metabolic activity brought about by mechanical and/or chemical signals. At this stage, the environment may be sealed out (in the case of coat-imposed dormancy), or the embryo may be under the influence of chemical messages that maintain dormancy. Chief among these chemical messengers is the plant hormone abscisic acid (ABA), a small organic molecule classified as an sesquiterpene and synthesized in plastids.

ABA in Dormancy

ABA plays a number of diverse roles in plants, and its name reflects its discovery based on its involvement in fruit abscission (detachment from the parent plant). ABA also plays a critical role in plant drought response, in part by regulating stomatal pore size. In seeds, ABA concentrations increase with embryo maturation due to its synthesis by the embryo, and results in several responses that promote dormancy. ABA promotes expression of seed storage proteins associated with seed maturity, known as Late Embryogenesis Abundant (LEA) proteins. ABA also promotes the synthesis of proteins associated with desiccation (drying) tolerance, known as heat-shock proteins (HSPs). In addition, ABA also prevents the premature germination of the embryo, known as vivipary.

Dormancy Release

The release of embryo dormancy is associated both with a decrease in ABA synthesis and concentration and an increase in the concentration of a second plant hormone, gibberellic acid (GA). As levels of ABA decrease, the inhibitory effects of this hormone begin to decline. At the same time, this decline promotes the synthesis of GA by the embryo. The increase in GA concentration actively promotes germination, with perhaps its most prominent influence occurring on the expression of several hydrolytic enzymes, including α- and β-amylase. α-amylase is known as an endoglucanase, meaning it hydrolyzes starch chains in mid-chain, while β-amylase is an exoglucanase, acting on the ends of the starch chain to release maltose units (a disaccharide of glucose). As starches are digested by these hydrolytic enzymes, the resulting sugars can be taken up by the embryo and used to fuel growth.


If high levels of ABA promote dormancy, and high levels of GA promote germination, how does the embryo “know” when to make the switch? There are a number of factors, both intrinsic and external, that influence this decision. External cues such as water availability and chilling exposure have been shown to influence germination, and internal cues related to seed maturation, known as afterripening, are also important in some species. Because plants are photosynthetic autotrophs, waiting to germinate until the light conditions were optimal would seem like an adaptive response, and in fact many plants use light as a cue to regulate germination. In particular, plants use the phytochrome photoreceptor to determine the quality of light available in the environment. Locations that are rich in high-energy light promote germination through the phytochrome signaling pathway, while locations predominated by shade inhibit germination.

Seed Structure


Seeds are amazing structures, capable of protecting an embryonic plant for hundreds of years in a state of suspended animation, awaiting the onset of favorable conditions to initiate germination. The seed represents a pause in the lifecycle of plants that is so effective, it conferred a major selective advantage on those species having it, the earliest of which arose in the late Devonian (417-354 Mya) and came to dominate by the early Mesozoic.

Seeds possess a number of improvements over spores that are adaptive, including a seed coat, multicellular embryonic seedling, and a significant reserve of energy. The thickness and material making up the seed coat varies from species to species and plays a role in the particular kind of dormancy experienced by a seed. The nature of the coat also influences the kinds of signals that are able to reach the embryo and release dormancy.

Seed Coat

Plants producing thick, lignified seed coats typically undergo coat-imposed dormancy, characterized by the physical exclusion of light and water, extremely limited gas exchange between the embryo and the environment, and the maintenance of high concentrations of the dormancy-promoting hormone abscisic acid. Some seeds having this kind of dormancy can endure for hundreds of years and remain viable [1]. These seeds are “hermetically sealed” packages, and must be broken open in order for germination to proceed. This breakdown may occur through the activity of animals (including exposure to digestive acids), exposure to freeze-thaw cycles, or other weathering processes.

Plants not having a thickened coat remain capable of dormancy, but rather than the physical restriction of exchange, dormancy arises from production of and response to particular chemical messages. In actual fact, both kinds of dormancy use chemical signaling to promote or release dormancy, but seeds with thickened coats have an additional mechanism for protecting the dormant state of the embryo.

Energy Supply

In addition to a seed coat, all seeds contain a supply of energy contributed by the parent. Depending on the kind of plant, this energy supply may take one of two different forms. In some plants the energy deposited by the parent remains in a large, starchy mass known as endosperm. Grasses (Family Poaceae) such as wheat (Triticum aestivum), maize (Zea mays), and rice (Oryza sativa) all maintain a prominent endosperm throughout seed maturation and dormancy. It is the endosperm, in fact, that we are eating when we eat grains like rice and wheat. In other plants, the endosperm may be absorbed fully by the developing embryo and stored in specialized seed leaves called cotyledons. Plants such as beans (Family Fabaceae) are representative of this strategy.


The embryo is the result of fertilization, or syngamy (fusion of gametes). Upon the fusion of the sperm nucleus with the egg nucleus inside the ovule, a single-celled zygote forms and begins developing into the embryo. As early as the first division of the zygote, polarity has already been established that will govern the apical-basal axis of the embryo. The embryo is fully dependent upon the parent plant for nutrition during seed maturation and early seedling establishment, until the seedling becomes photosynthetic and therefore autotrophic (self-feeding). Before this, the embryonic plant is in fact heterotrophic, benefiting from the investment of photosynthate from its parent.

Shen-Miller et al. (1995) Exceptional Seed Longevity and Robust Growth: Ancient Sacred Lotus from China. American Journal of Botany 82: 1367-1380.