Properties of meristems

Meristems contain a population of cells with characteristics of stem cells; cell division serves to constantly replenish the meristem and to provide cells that will differentiate into plant organs and tissues. Unlike most examples of stem cells in animals, where the potency of the differentiating daughter is restricted, cells produced by plant meristems have the capacity to differentiate as any cell type. Thus some prefer to think of meristems as populations of embryonic cells because the cells produced go through the entire process of organogenesis, pattern formation, histogenesis and differentiation.

Vegetative meristems are indeterminate. That is they can theoretically grow indefinitely. In plants that normally produce a relatively fixed number of nodes, if the meristem is removed and allowed to reform a plant in culture, the plant will have the normal number of nodes. Thus node number is a property of the whole plant and not intrinsic to the meristem.

Vegetative meristems are highly regulative. If portions are surgically removed, the remainder will reorganize into a functioning meristem.

The activity of vegetative meristems is repetitive, often described as iterative or meristic (from mer meaning unit as in polymer). The meristem produces modular units consisting of a lateral organ (leaf), axillary bud, node and subtending internode. Each unit is called a phytomer. Thus primary shoot growth involves the repetitive addition of stem segments and associated leaves to the end of the shoot.

Meristem structure

SAMs are usually dome shaped and have a layered structure described as a tunica and corpus. The tunica consists usually of 2 cell layers (often only 1 in monocots) where cell division occurs primarily in the anticlinal plane (perpendicular to the surface).The corpus is the mass of cells in the central part of the meristem and cell divisions occur in all planes. An alternative nomenclature is to number the layers from the outside in. Thus plants with a 2 cell layered tunica would have an L1 layer on the outside, L3 layer in the center and an L2 layer in between, with both the L1 and L2 being part of the tunica and the L3 comprising the corpus. Monocots such as maize that have only a single layer tunica would have an outer L1 layer and an inner L2 layer with nothing in between.

SAMs also show cytological zonations independent of the layered structure. There is a central zone that stains lightly with histological stains. The central zone includes both tunica and corpus cells. The peripheral zone is a ring of densely staining cells that surround the central zone and also includes both tunica and corpus. Below the central zone is an area called the rib meristem. Several approaches have shown a correlation in the mitotic activity with the zonation. Mitotic activity is most prevalent in the peripheral region as shown by incorporation of tritiated thymidine which reflects DNA replication and therefore mitotic activity.

The central zone is the region of the apical initials. These divide infrequently and occasionally contribute cells to the rapidly dividing peripheral zone. Because most genetic mistakes are generated during mitosis, this is probably a mechanism to minimize the occurrence of genetic mistakes in the permanent cell population of the meristem.

Initiation and maintenance of meristems

Initiation of the shoot meristem in the embryo requires the action of several genes that were identified in genetic screens for mutants that lack a SAM. One factor is a pair of genes called CUP-SHAPED COTYLEDONS (CUC)1 and 2. These are duplicate factors. That is that there are two different genes that function redundantly and both have to be mutated to see a mutant phenotype. The phenotype consists of both cotyledons forming in a continuous fused cup-shaped structure with no SAM. Occasionally, one can get a shoot to form in tissue culture and then growth is fairly normal up to flowering. This indicates that the CUC genes are required for SAM initiation but not maintenance.

Mutations in the SHOOT MERISTEMLESS (STM) also result in embryos without a SAM. Occasionally, in weak stm alleles, adventitious shoots will form but the shoots progressively lose their meristem indicating that STM is required for both the initiation and maintenance of SAMs. STM encodes a homeodomain transcription factor.

Maintenance of shoot apical meristems requires regulating the balance between cell proliferation and differentiation. Several genes have been identified that regulate this balance. As mentioned, STM is required to maintain the SAM and in mutants the SAM dwindles away because of either too rapid cell differentiation or insufficient cell proliferation. Accordingly, the proposed function of STM is to either promote proliferation (ie promote the meristematic state) or to inhibit differentiation. Consistent with this, ectopic expression of STM-like genes, such as the maize KNOTTED gene, in leaves causes the formation of ectopic meristems (see Howell, fig. 5.6). Another gene called WUSHEL (WUS) has a similar function (ie, similar mutant phenotype) and also encodes a homeodomain transcription factor.

Three genes called CLAVATA (CLV)1,2 and 3 have the opposite effect in that mutations cause an overproliferation of cells in the SAM (the SAM gets too big). Therefore the function of these genes is to restrict cell proliferation or to promote cell differentiation. CLV1 is a receptor kinase and CLV2 is another receptor-like protein. These 2 are proposed to form a heterodimeric receptor complex. CLV3 is a small protein that is predicted to be secreted and is hypothesized to function as the activating signal ligand for the CLV1/2 receptor complex. CLV1 is expressed mostly in the corpus region of the SAM and CLV3 is expressed predominantly in the tunica region of the SAM, highlighting the interaction among layers of the SAM. Because cells in both the tunica and corpus proliferate abnormally in clv1 mutants, it is concluded that CLV1 acts non-autonomously. It is possible that activation of the CLV1 receptor results in the production of a secondary signal that diffuses throughout the meristem.

The balance between the activity of the proliferation promoting (differentiation inhibiting) and the proliferation inhibiting (differentiation promoting) pathways is required for the long-term function of the SAM. The CLV and STM pathways are antagonistic to each other because clv and stm mutants partially suppress one another. This also indicates that these represent independent pathways because each still functions in the absence of the other. On the other hand, the WUSHEL gene acts downstream of CLV because clv / wus double mutants look just like wus. The CLV pathway acts to inhibit the expression of WUS which acts to promote cell proliferation. In clv mutants there is nothing to restrict WUS, resulting in overproliferation. However, if wus is mutant, then it doesn’t matter whether CLV is around to restrict it or not.

Organ initiation

Leaf initiation involves an increased rate of cell division on the flank of the meristem. This involves both tunica and corpus cells. Cell divisions in the L1 remain anticlinal whereas inner layers divide both anticlinally and periclinally. The proliferation of cells leads to a bulge on the flank of the meristem called a leaf buttress. Localized apical growth in the leaf buttress generates a leaf primordium. Apical leaf growth continues until the primordium is approximately 1mm long (the length is variable among species) at which point the leaf pattern of cell division begins.

The time between the initiation of two leaves is called a plastochron. Primordia are referred to by counting from the youngest to oldest. Thus the 4^th youngest primordium would be referred to as a plastochron 4 leaf because it is 4 plastochrons old.

Patterns of organ initiation (phyllotaxy)

Different plants have characteristic arrangements of leaves such as alternate, opposite (decussate), spiral or whorled. The arrangement of leaves on the plant is called the phyllotaxy and is determined by the pattern of leaf initiation on the meristem. In plants with opposite or alternate arrangement, leaves occur in files on the stem called orthostichies. Plants with spiral leaf arrangement have an arrangement that can be described with 2 opposite sets of helical rows called contact parastichies. Phyllotaxy is described by counting the spirals in each direction. For example, potato has 2 clockwise parastichies and 3 counterclockwise. This phyllotaxy is described as 2+3. Phyllotaxy usually consists of consecutive numbers of the Fibonacci series: 1,1,2,3,5,8 where each successive number is derived by adding the 2 preceeding numbers.

A divergence angle between leaf primordia of 137^o is nearly constant among plants with spiral leaf arrangement. The generation of the different phyllotaxies is a property of differences in the relative rates of growth and leaf initiation in the meristem and meristem size. This will affect which primordia are in contact with one another.

Control of organ initiation patterns

Three major hypotheses exist to explain how organ initiation patterns are generated. These are not mutually exclusive hypotheses, although proponents argue about them as if they were.

Inhibitory field hypothesis:

One way to explain the pattern of organ initiation is that organ primordia produce an inhibitory field that prevents organ initiation within a certain proximity. Surgical manipulations in which the position of organ initiation was assessed following removal of a primordium are generally consistent with this theory. Subsequent organs that would normally have contacted the removed primordium initiated at positions closer than normal to the missing primordium. A corollary is that the apical dome should also produce an inhibitory field.

Auxin appears important for leaf initiation. Application of auxin transport inhibitor to an apex results in the simultaneous initiation of primordia in a continuous collar surrounding the apex. If the auxin transport inhibitor is spotted on primordia, they occupy a larger position on the meristem. An inhibitor of auxin action spotted onto primordia caused them to occupy a smaller area. Both these treatments resulted in altered phyllotaxy. The results suggest that a localized high auxin concentration promotes organ initiation and determines the size of the field occupied by the primordium. The inability to transport auxin away from the apex results in a uniformly high concentration causing the continuous collar. The inability to transport auxin away from a primordium results in a higher localized concentration and larger field.

Biophysical hypothesis:

According to biophysical explanations of phyllotaxy, patterns of stress and reinforcement in the surface layer of the apex determine the pattern of organ initiation. The inner tissues are hypothesized to provide the driving pressure, much like the air in a balloon. Polarized light microscopy shows a correlation between phyllotaxy and cellulose microfibril orientation in the apex surface.Results of the surgical and auxin experiments are not incompatible with this model because the surgery might have altered the pattern of physical stress in the apex and the auxin may have affected reinforcement by cell wall loosening.

The protein expansin is also able to induce cell wall loosening. Expansin coated beads were placed on meristems in a position other than the site of the next predicted primordium initial. This treatment inhibited formation of the primordium that normally would have formed and allowed the formation of a primordium in the ectopic location. Ie. the pattern of phyllotaxy was altered. The new phyllotaxy was propagated in subsequent primordia. This supports the role of biophysical properties of the surface in organ initiation. Expansin proteins are expressed at sites of organ initiation in the SAM, supporting the possible role of expansin in normal organ initiation but how the pattern of protein accumulation is regulated is not yet known.

Genetic control:

There are several genes known to be important for meristem function. The knotted gene of maize and STM gene of Arabidopsis encode homeodomain proteins that are expressed in the apical meristem. Expression patterns are consistent with the proposed function of promoting meristem function/inhibiting differentiation. Both are expressed throughout the meristem except in organ primordia. Loss of expression precedes visible histological appearance of the primordium. clavata mutants of arabidopsis have abnormally large meristems with altered phyllotaxy. Specifically, the central zone is enlarged and more than the normal number of organs are formed (this is most apparent in flowers). This is consistent with inhibitory fields controlling primordia initiation. Another gene of maize called abphyl1 also has an enlarged meristem with altered phyllotaxy. Instead of forming leaves alternately, mutants form leaves oppositely. The expression pattern of knotted is altered in accordance with the pattern of leaf initiation but the identity of abphyl1 is not yet known.

Organ determination

Determination of leaf primordia was studied in ferns by explanting primordia to sterile culture free of hormones. Plastochron 2 primordia usually developed shoots (ie. they still had a meristem identity. By plastochron 8, primordia are 1 mm and developed into normal looking leaves (although smaller than usual). The primordia acquired leaf identity and contained the information to develop autonomously.

Leaves are determinate organs. That is they grow to a specified size and no further. Thus along with the acquisition of leaf identity is the loss of indeterminate growth.

Clonal analysis 1. fate of meristem cells in the shoot.

Nomenclature:

chimera—a plant composed of genetically different cells (ie. a genetic mosaic)

periclinal chimera—a chimera in which entire cell layers of the apical meristem are genetically different from one another.Albino mutations are commonly used to mark cell lineages. These chimeras are described with G and W according to the cell layer. Thus chimeras with a white L1 and green L2 and L3 would be denoted W-G-G, white L2 would be G-W-G, white L2 and L3, G-W-W etc.

mericlinal or sectorial chimera—chimeras that do not occupy the whole circumference of the meristem

Genetically marking cells within meristems allows the cells to be followed and their fates revealed. Such lineage studies have revealed several things about meristems. First, periclinal chimeras are fairly stable indicating that the tunica corpus arrangement of cells in the meristem represent stable cell lineages. This allows propagation of several variegated horticultural plants.

Rarely, cells from one apical layer will enter another layer. In ivy a clonal analysis was done by examining rare changes in the variegation pattern. The few permanent changes observed affected a quarter of the shoot indicating that each apical layer contained 4 apical initial cells. Most changes were ephemeral and the longest ephemeral changes occupied 8 nodes indicating that one daughter of the apical initials contributed to 8 nodes worth of growth.

Fate maps have been generated for the apical meristem in the embryo of several plants by using ionizing radiation on seeds to induce albino sectors. The general pattern observed is that a few cells at the center of the meristem contribute to many nodes at the top of the plant, while many cells on the sides of the meristem contribute to few nodes at the bottom of the plant.

Clonal analysis 2. fate of meristem cells in lateral organs.

Examination of the pattern of green tissues on leaves of periclinal chimeras reveals the fate of each cell layer in the organ. This analysis shows that L1 cells contribute the epidermis and in some plants, tissues around the leaf margin. L2 cells contribute most of the mesophyll tissue and L3 cells contribute some of the mesophyll in the central region of the leaf near the midvein. In G-W-G chimeras, the occasional invasion of epidermally derived cells into the mesophyll is easy to detect. Single green cells have been observed in otherwise albino areas of mesophyll. These cells differentiate as mesophyll cells indicating that cell fate is not determined until after the last cell division and emphasizing the independence of cell lineage and cell differentiation.

A study was done in maize where sectors were induced at the time of leaf initiation and the relative contribution of the sectors were analyzed in the resultant leaf. By analyzing the fractional contribution of the sectors to the leaf, the number of apical meristem cells that form the primordium could be estimated. Epidermal sectors either occupied 2/3 of the top side, 2/3 of the bottom side or 1/3 the bottom and top sides at the tip. This indicated that 3 cells in the longitudinal dimension contributed to leaf formation. Sectors occupied either the epidermis or mesophyll indicating that at least 2 cells in depth contributed to the leaf. Most sectors occupied 1/40^th the width of the leaf indicating that the primordium was 40 cells wide. Thus roughly 240 cells contributed to the formation of a maize leaf.

Development in the meristem is highly regulative

The late stage at which cell fate is determined (ie. after the last cell division) and the ability of cells to invade different cell layers and differentiate according to their final position indicate a highly regulative development. This can be further demonstrated by inducing periclinal chimeras in which clones have different growth characteristics. The relative contributions of different cell layers to leaves can be altered in various chimeras but the overall size and shape of the meristem or leaves does not change.

Branching

There are two types of branching patterns in plants: terminal and lateral.

Terminal branching involves the separation of the apical meristem into 2, which then both continue growing as a shoot branch. This mechanism is common in lower plants but rare in angiosperms. How the single integrated growing center is divided is not understood. Experimental manipulations such as bisecting the meristem can artificially induce terminal branching, but these do not appear to mimic any naturally occurring events. They do speak to the capacity of isolated meristem segments to reorganize into complete meristems.

Lateral branching in seed plants occurs mainly by growth of axillary buds. When leaf primordia are initiated, a small group of cells in their axils remain small and unvacuolated. These are referred to as “detached meristems” because they retain meristematic characteristics and do not differentiate as the cells around them do, but they become spatially separated from the apical meristem. As the shoot grows and the detached meristems attain a distance from the apex, they organize into a shoot meristem with several leaf primordia (a bud). At this point they arrest because of apical dominance (the shoot apical meristem inhibits growth of the axillary meristems by producing the hormone auxin). In bushy plants the arrest is temporary until the main shoot has grown beyond the inhibitory range of the apex. In unbranched plants the arrest is permanent unless the apical meristem is removed. Upon release from apical inhibition, the lateral bud begins growth as a branch shoot.