A developmental gradient is apparent at the growing tip of roots. This gradient can be subdivided into 3 zones. The apical tip is the meristem or zone of cell division. The next zone proximal to the meristem is the zone of elongation where cell division ceases and there is rapid cell growth by elongation. Then comes the zone of differentiation or specialization, where cells assume their final fate. The zone of differentiation is made obvious by the appearance of root hairs in the epidermis.
The outer layer of cells is the epidermis, next layer is the cortex, followed by the endodermis, pericycle and vasculature. Near the root tip, there is also a layer of lateral root cap cells outside the epidermis. In arabidopsis, each of these tissues corresponds to a single layer of cells.
The Root Apical Meristem (RAM)
Primary root growth occurs at the root apical meristem (RAM). Unlike the SAM, the RAM produces cells in two directions. The RAM produces a cap of tissue called the root cap, which covers the distal tip of roots. The root cap protects the root tip as it grows through the soil. Cells are continuously sloughed off the outer surface of the root cap. The RAM also produces cells proximally that contribute to the root proper, but unlike the SAM, the RAM produces no lateral appendages.
The RAM contains the only cells in the plant embryo that are derived from the suspensor rather than the embryo proper. Part of the RAM is derived from the embryo proper but the quiescent center and columnella initials (see below) are derived from the very last cell of the suspensor, called the hypophesis.
In many root meristems, cell files can be traced back to a small group of initials. Often these initials are organized in a tiered arrangement, with each tier giving rise to particular tissues. In radish, tier 1 produces root cap and epidermis, tier 2 cortex and tier 3 vasculature. In maize, tier 1 produces only root cap, tier 2 epidermis and cortex, tier 3 vasculature. Other plants show no tiered arrangement or only a single initial layer. Many lower plants such as ferns have a single large pyramidal cell called the apical cell. The apical cell alternates cleavages along its four faces and is the ultimate source of all cells in the meristem.
The center of the RAM is occupied by a quiescent center which has low mitotic activity. The quiescent center is most apparent in actively growing roots and is lost during dormancy, carbohydrate starvation or root cap removal. This is the same region of the meristem to which cell files trace and was postulated to contain the root initials. Evidence suggests the quiescent center does function as the zone of initials. Colchicine induces polyploidy in any cells undergoing mitosis at the time of treatment. In actively growing roots treated with colchicine, sectors of polyploid cells were transient indicating that no initial cells were dividing at the time of treatment. If the quiescent center is eliminated by removal of the root cap, prior to colchicine treatment, then stable sectors of polyploid cells are produced and persist after re-establishment of the quiescent center. Thus infrequent division of initial cells in the quiescent center is the source of cells for the RAM. More recent cell lineage experiments have used transposon activation of a GUS reporter to mark root cell lineages, leading to the same conclusions.
Surgical manipulations have provided information about RAM organization. Quiescent centers surgically explanted to sterile culture regenerated a meristem and grew as a root. Bisected RAMs formed 2 meristems and roots. These experiments demonstrate the organizing capacity for RAMs. Experiments with maize which has a highly tiered meristem suggest the layered structure is not required for normal function. Portions of the maize RAM were removed by glancing incisions and when the RAM regenerated, the layered arrangement was no longer present on the cut side, but was still apparent on the undamaged side. Although the structural arrangement of cells differed from one side to the other, the meristem still produced a normal root. The layered structure may therefore reflect “status quo” patterns of cell division but not have functional significance in the normal production of root tissues.
Arabidopsis roots are extremely small and simple: the radial pattern consists of concentric arrangements of 6 different tissues, each a single cell layer thick. This allows every cell to be traced back to a specific progenitor in the meristem. All cells trace back to a small group of cells called the promeristem (see handout) and the pattern of cell divisions in the promeristem is invariant. Fate mapping showed that the central cells and columnella root cap cells of the embryonic RAM are derived from the basal cell formed by the first zygotic division, while the remainder of the promeristem is derived from the apical cell.
Although cell divisions in the arabidopsis RAM typically follow a set pattern, generating relatively invariant cell lineages, laser ablation studies indicate that position rather than lineage is the important factor for determining cell fate (identity). For example, if a cortical initial is ablated, a pericycle cell will invade the voided space, assume the pattern of cell division characteristic of a cortical initial and give rise to cortical and endodermal derivatives. Thus the cell changes fate from pericycle to cortical initial when it’s position is altered.
These studies also demonstrated that the differentiated cells proximal to the RAM influence the fate of cells in the RAM. The cortical/endodermal initial undergoes a transverse division after which the daughter undergoes a periclinal division to give rise to the endodermis and cortex. If three adjacent cortical initial daughter cells were ablated, such that a cortical initial was no longer in contact with any daughter cell, the initial still divided to produce a daughter but now the daughter did not undergo the typical asymmetric periclinal division to generate the cortical and endodermal cell files. Therefore, the more mature, differentiated cells direct the pattern of division and differentiation of the initial cells (van den Berg, 1995).
Two mutants show the same phenotype: endodermis and cortex are replaced by a single cell layer. In the short root mutant the single layer has characteristics of cortex thus shr is required to specify endodermal identity. In contrast, scarecrow has both endodermal and cortical characteristics in the single layer and thus appears to be involved in specifying the cell division that generates the two layers.
The quiescent center regulates the differentiation of neighboring cells. If one or 2 QC cells were ablated, the columnella initials that were no longer in direct contact with a QC cell ceased dividing and underwent differentiation as a columnella cell. Similarly, cortical initial cells that were no longer in direct contact with the a QC cell behaved as cortical daughter cells and underwent an asymmetric division to generate cortical and endodermal cell files. Therefore, direct contact with the QC inhibits the differentiation of initial cells in the RAM (van den Berg, 1997).
An GUS reporter for auxin concentration shows a concentration of auxin just at the distal end of the vasculature, just proximal to the RAM. Disruption of this localized accumulation by auxin transport inhibitors, exogenous auxin application or mutations in genes involved with auxin response, disrupts the organization of cell types in the root apex, as visualized by promoter trap cell markers. Thus the proper localization and perception of this auxin maximum are important for organizing the RAM (Sabatini, 1999).
Root hairs are filamentous structures produced by a subset of cells on the root epidermis. They function to increase the surface area of the root, allowing more efficient water and nutrient uptake. The root epidermis consists of elongated cells of 2 cell types: hair cells and hairless cells. Positional cues specify the pattern of root hair development. Arabidopsis roots are particularly well suited for studying cell patterning. The root is small, with a 1 cell layered cortex, and a very regular pattern of cell division in the RAM that allows the lineage of each cell to be known. The cortex consists of an invariant ring of 8 cells. The epidermis consists of a variable number of cells but there is invariably 8 files of root hair cells. Root hairs always form on epidermal cells positioned over the radial cell wall between cortical cells. The control of cell pattern will be considered in more detail in a later lecture.
Root hair cells can be cytologically distinguished by their dense cytoplasms (delayed vacuolation) early in development, near the onset of elongation; thus cell fate has been specified prior to elongation. The root hair first becomes visible as a swelling at the apical end of the elongate epidermal cell. The protuberance then grows into a root hair by tip growth, similar to pollen tubes. It requires a Ca++ gradient, golgi vesicle transport and vesicle fusion with the plasma membrane.
Like shoot branching, root branching can be either terminal or lateral, with the terminal mode being more common in lower plants and lateral much more common in angiosperms. Terminal branching involves the division of the RAM into 2 with the subsequent production of 2 roots. Lateral branching is different in roots than in shoots. Lateral roots initiate from internal cells of the pericycle. Initiation occurs in the late cell elongation/early cell differentiation zone, in pericycle cells that are partially to fully differentiated. Thus there is no detached meristem. A small group of pericycle cells reorient their axis of polarity to the radial dimension and begin growing and dividing to form a mound of cells. With continued growth and division, the mound of cells becomes organized into a RAM with root cap, while still within the tissues of the main root. Continued growth allows the lateral root to penetrate the endodermis, cortex and epidermis, finally reaching the exterior of the parent root.
Auxin and cytokinins are key factors in determining lateral root initiation. Exogenous auxin induces a extra lateral roots in a dose dependent manner (ie. more auxin, more lateral roots). Conversely, many auxin mutants show a paucity of lateral roots. Environment also influences lateral root development. Lateral roots in nitrogen deficient environments will respond to applied NO3- by elongating. This requires the function of a myb transcription factor called ANR1. Lateral roots of antisense ANR1 plants do not respond to nitrate. This also demonstrates that this is a specific response to the nitrate stimulus and not simply a nutritional response because the antisense plants are otherwise healthy.
Several soil nutrients can alter root hair development (Reviewed in López-Bucio et al, 2003). Fe or P deficiencies both induce more epidermal cells to differentiate as root hairs, and both induce root hairs to elongate more than normal. These two pathways appear independent. The Fe pathway appears to function through the ethylene and auxin pathways because ethylene and auxin mutants show altered responses to Fe deficiency. P appears to be ethylene and auxin independent. The increased root hairs increases the surface area of roots, increasing their capacity to absorb limited nutrients.
Several nutrients can also alter root architecture by altering lateral root formation or growth, or by altering primary root growth. High nitrate inhibits lateral root elongation if the root system is uniformly exposed. However if only a portion of the root system experiences high nitrate while the rest experiences deficiency, the section with high nitrate will show elongated lateral roots. A MADS box transcription factor, ANR1 is induced by local high nitrate and is required for the root architecture response.
Phosphate deficiency induces the formation of lateral roots and inhibits root elongation. The result is a dense, highly branched root system. This is compounded by the effect on root hairs. In addition, expression of phosphate transporter genes and other physiological changes result in a root system highly adapted for efficient uptake of P. The effects on root growth are brought on by inhibition of the cell cycle and by low auxin concentrations in the root apical meristems.
Sulfate deficiency also increases lateral root density. The NIT3 (nitrilase3) gene is induced and thought to increase auxin synthesis.
Several lines of evidence suggest the nutrient ions may act directly as signaling molecules. Mutants in nitrate metabolism still show the normal response to Nitrate. Root systems on plants with adequate phosphate show the classic P starvation phenotype in localized areas of deficiency. Thus the changes in root architecture are not secondary effects of altered metabolism, but appear to be primary effects regulated by the ions themselves.
López-Bucio, J., Cruz-Ramirez, A. and Herrera-Estrella, L. (2003) The role of nutrient availability in regulating root architecture. Curr Opin Plant Biol 6: 280-287
Sabatini S, Beis D, Wolkenfelt H, Murfett J, Guilfoyle T, Malamy J, Benfey P, Leyser O, Bechtold N, Weisbeek P, Scheres B (1999) An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell 99: 463-72
van den Berg C, Willemsen V, Hage W, Weisbeek P, Scheres B (1995) Cell fate in the Arabidopsis root meristem determined by directional signalling. Nature 378: 62-5.
van den Berg C, Willemsen V, Hendriks G, Weisbeek P, Scheres B (1997) Short-range control of cell differentiation in the Arabidopsis root meristem. Nature 390: 287-9.