Root apical organization inArabidopsis thaliana 1. Root cap and protoderm

Summary We investigated the development of the root cap and protoderm inArabidopsis thaliana root tips.A. Thaliana roots have closed apical organization with the peripheral root cap, columella root cap and protoderm developing from the dermatogen/calyptrogen histogen. The columella root cap arises from columella initials. The initials for the peripheral root cap and protoderm are arranged in a collar and the initiation event for these cells occurs in a sequential pattern that is coordinated with the columella initials. The resulting root cap appears as a series of interconnected spiraling cones. The protoderm, in three-dimensions, is a cylinder composed of cell files made up of packets of cells. The number of cell files within the protoderm cylinder increases as the root ages from one to two weeks. The coordinated division sequence of the dermatogen/calyptrogen and the increase in the number of protoderm cell files are both features of post-embryonic development within the primary root meristem.Abbreviations RCP root cap/protoderm - CI columella initial - PI protoderm initial     

THE ULTRASTRUCTURE OF THE QUIESCENT CENTER IN THE APEX OF CULTURED ROOTS OF CONVOLVULUS ARVENSIS L.

Cultured roots of the common bindweed, Convolvulus arvensis L. growing at the rate of 15–30 mm/day in sterile nutrient medium were fixed for electron microscopic analysis. The ultrastructure of the quiescent center, the initials of the ground meristem, and the initials of the procambium were studied in order to determine whether sequential structural changes could be correlated with models for specifying the mechanisms by which cell differentiation and cell division might be controlled. The differentiation of cells in the root proper occurs very gradually in linear files from the site of the quiescent center proximally into the different tissue regions. Major structural changes, such as the orientation and subsequent elongation of cells along the longitudinal axis of the root and cell wall changes, indicate that the control of differentiation and perhaps cell division occurs in radial gradients outwardly from the quiescent center.     

PHOSPHATASE ACTIVITY AND CELLULAR DIFFERENTIATION IN PHLEUM ROOT MERISTEM

Using 9 different organic phosphate substrates as alternatives in a standardized 5′-nucleotidase histochemical test system, enzyme activity patterns were recorded for timothy grass root epidermis. At least 4 different phosphatases were distinguished on the bases of substrate specificity, reaction rate, tissue distribution, and response to inhibitors. Except with adenosine-3′-monophosphate, all activities were restricted to the 300-mμ-long root tip meristem. These enzyme activities were associated with the earliest phases of differentiation of the epidermal hair and hairless cell initials. The distribution of activities was not associated with the same cell type in each part of the meristem. Little activity was found with most substrates in the undifferentiated cells of the 0-100μ zone; alternating active hairless and inactive hair cell initials predominated in the 100-200μ segment; and active hair–inactive hairless sister cells formed the principal pattern in the 200-300μ segment of the meristem. The data showed that a particular enzyme activity was associated with a specific cell type only in relation to that cell's position along the differentiation gradient of the entire tissue. But, within a meristem segment, a specific cell type might act differently from its neighbors, depending on its mitotic capacity. This complex of physiological dependence and independence of a cell type on tissue ontogeny was cited as a characteristic of the phenomenon of cellular differentiation superimposed on tissue differentiation gradients.     

Cell division patterns of the protoderm and root cap in the “closed” root apical meristem ofArabidopsis thaliana

Summary The peripheral root cap and protoderm inArabidopsis thaliana are organized into modular packets of cells derived from formative T-divisions of the root cap/protoderm (RCP) initials and subsequent proliferative divisions of their daughter cells. Each module consists of protoderm and peripheral root cap packets derived from the same periclinal T-division event of an RCP initial. Anatomical analyses are used to interpret the history of extensively coordinated cell divisions producing this modular construction. Within a given layer of root cap, the columella and RCP initials divided in a centrifugal sequence from the innermost columella initials toward the RCP initials. All RCP initials in the lineages around the circumference of the root divided nearly simultaneously in waves to form one module prior to the next wave of initial divisions forming a younger module. The peripheral root cap and protoderm packets within each module completed four rounds of proliferative divisions in the axial plane to produce, on average, 16 cells per packet in the basalmost modules in axial view. Peripheral root cap and protoderm cells predominantly in the T-type (trichoblast) lineages also underwent radial divisions as they were displaced basipetally. The regularity in the cellular pattern within the modules suggests a timing mechanism controlling highly coordinated cell division in the initials and their daughter cells.Abbreviations RAM root apical meristem - RCP root cap protoderm - prc peripheral root cap     

NUCLEOLAR VARIATION DURING DIFFERENTIATION OF PHLEUM ROOT EPIDERMIS

Striking differences in nucleolar volume were found between trichoblasts and hairless initials all along the differentiation gradient of the tissue. The larger nucleoli of the trichoblasts were evident from the onset of their differentiation in the meristem and remained so throughout the growing 1000μ of epidermis. At the same time, nucleoli of the alternating, hairless initials rapidly became reduced in volume and virtually disappeared in maturing cells. Nuclear volume was relatively constant throughout the epidermis, so that nuclear:nucleolar volume ratios clearly indicated the nucleolar differences between the shorter trichoblasts and longer hairless initials. Along with the first signs of nucleolar volume difference in the meristem, there were higher concentrations of RNA and ribonucleoprotein in nucleoli and cytoplasm of trichoblasts compared with hairless initials. Although these chemical differences occurred principally in meristem cells, protein content was much higher in trichoblasts than in the alternating hairless initials of the enlargement zone, 300–500μ from the root tip apex. There was no essential difference in protein content between the 2 cell types in the meristem or in older enlarging cells. The data suggested that the initial increase in nucleolar volume and content of the meristem trichoblasts led to their increased protein content and enzyme activities during the enlargement phase of their differentiation. The sharp reduction in nucleoli of the hairless initials, at the same time, led to their generally lower metabolism during tissue differentiation.     

Meristem Initial Cells in Irradiated Roots of Vicia faba: With one Figure in the Text

Beans (Vicia faba) were germinated for 24 hours and irradiatedwith X-rays. Primary roots were fixed after 9, 11, and 21 days.Aberrant chromosome complements, the result of chromosome changesinduced by irradiation, were present. They were used as cellmarkers to estimate the number of cell types present in themeristem initial cells of the growing root and the number ofprimordium initial cells from which root regeneration occurred.Up to 9 cell types occur as meristem initials. From the relativefrequencies of the different cell types, it is estimated thatthere are at least 32 actual meristem initial cells in regeneratingirradiated primary roots. This result is compatible with observationsmade on normal roots. The chimaerical nature of the regeneratingroot apparently does not interfere with the normal organizationof the meristem, but it serves to reveal what part of the organizationis.     

Comparative anatomy of root meristem and root cap in some species of Podostemaceae and the evolution of root dorsiventrality

In the unusual aquatic Podostemaceae, the root is the leading organ of the plant body and is variously compressed and submerged as it adheres to rock surfaces in rapid water. In an anatomical comparison of the root apical meristems and root caps of 33 species that represent the major lineages of the family, the dorsiventrality of root meristems varied and was classified into four patterns: (1) The root cap is produced outward from a nearly radially symmetrical meristem. (2) The meristem and root cap are markedly dorsiventral; the outermost cells of the hood-shaped cap are acroscopic derivatives from bifacial initials on the ventral side, while the pattern on the dorsal side is similar to pattern 1. (3) Bifacial initials are on both the dorsal and ventral sides. (4) No root cap is present. An evolutionary polarity may be evident from pattern 1 to 2 and then to 3. Pattern 2 arose in the early evolution of the subfamily Podostemoideae and subsequently, pattern 3 arose in species with crustose roots, while the least specialized pattern 1 is retained in Tristichoideae and Weddellinoideae. Pattern 4 characterized by caplessness may have appeared recurrently in Tristichoideae and Podostemoideae. These evolutionary changes in the meristem preceded the specialization of external root morphologies.     

Apical organization and maturation of the cortex and vascular cylinder inArabidopsis thaliana (Brassicaceae) roots

Developmental and physiological studies of roots are frequently limited to a post-germination stage. In Arabidopsis, a developmental change in the root meristem architecture during plant ontogenesis has not previously been studied and is addressed presently. Arabidopsis thaliana have closed root apical organization, in which all cell file lineages connect directly to one of three distinct initial tiers. The root meristem organization is dynamic and changes as the root ages from 1 to 4 wk post-germination. During the ontogeny of the root, the number of cells within the root apical meristem (RAM) increases and then decreases due to changes in the number of cortical layers and number of cell files within a central cylinder. The architecture of the initial tiers also changes as the root meristem ages. Included in the RAM's ontogeny is a pattern associated with the periclinal divisions that give rise to the middle cortex and endodermis; the three-dimensional arrangement of periclinally dividing derivative cells resembles one gyre of a helix. Four- or 5-wk-old roots exhibit a disorganized array of vacuolated initial cells that are a manifestation of the determinate nature of the meristem. Vascular cambium is formed via coordinated divisions of vascular parenchyma and pericycle cells. The phellogen is the last meristem to complete its development, and it is derived from pericycle cells that delineate the outer boundary of the root.     

DEVELOPMENTAL ANATOMY IN ROOTS OF IPOMOEA PURPUREA. I. RADICLE AND PRIMARY ROOT

The developmental anatomy of the primary root of Ipomoea purpurea was studied at several growth stages, beginning with the radicle. The radicle is generally composed of three superimposed tiers of initials, which produce the vascular cylinder, cortex, and columella; and a peripheral band of lateral rootcap-epidermal initials. The radicular cortex contains 16–19 immature laticifers; none of the tissue regions in the radicle contains mature cells. Following germination and during the first 2–3 cm growth of the primary root the apical meristem and its derivative tissues undergo a series of modifications. Root apical diameter decreases as cells in lateral portions of the rootcap elongate; meanwhile, the columella enlarges vertically. The relationship between cortical and columellar initials changes as fewer mitoses occur in the former while the latter remain active. In longer roots the columellar initials are directly in contact with the vascular initials. Cortical size diminishes during early root growth as cortical laticifers and their associated cells cease to be produced by the outer cortical initials and ground meristem. Early procambium, at the level of vascular pattern initiation, decreases in diameter by cellular reorientation, and the vascular cylinder decreases in overall diameter although the tetrarch pattern remains unchanged.     

ONTOGENETIC REORGANIZATION OF THE ROOT MERISTEM IN THE COMPOSITAE

The organization of the root meristem in selected Compositae was investigated to determine whether changes in the pattern of cell arrangement occurred during root growth in species other than Helianthus annuus. Embryonic, short, and long primary roots of one species of each of twelve genera were prepared for microscopic examination. Additional intermediate growth stages were prepared for Echinacea pallida. The meristem of embryonic roots showed layers of initials typical for dicotyledons. The meristem in many of the short roots of eight species was reorganized by the development of a secondary columella. The long roots showed patterns similar to the embryonic roots. In three species which maintained closed meristems, two layers of cortical initials were common in the embryonic root, and as a general trend, a single layer of cortical initials became more common during root elongation. The cellular changes that resulted in the initiation of a secondary columella are characterized by the conversion of cortical initials to secondary columella initials by a shift in their plane of cell division. It is proposed that the size and shape of the quiescent center changes as the conversion takes place. No intermediate stages were observed which could account for the reduction of two layers of cortical initials to one layer.     

Clonal analysis of the Arabidopsis root confirms that position, not lineage, determines cell fate

 The cellular organization of the Arabidopsis thaliana (L.) Heynh. root meristem suggests that a regular pattern of cell divisions occurs in the root tip. Deviations from this pattern of division might be expected to disrupt the organization of cells and tissues in the root. A clonal analysis of the 3-d-old primary root meristem was carried out to determine if there is variability in division patterns, and if so to discover their effect on cellular organization in the root. Clones induced in the seedling meristem largely confirmed the predicted pattern of cell divisions. However, the cellular initials that normally give rise to the different cell files in the root were shown to exhibit some instability. For example, it was calculated that a lateral root cap/epidermal initial is displaced every 13 d. Furthermore, the existence of large marked clones that included more than two adjacent cell layers suggests that intrusive growth followed by cell division may occur at low frequency, perhaps in response to local cell deaths in the meristem. These findings support the view that even in plant organs with stereotypical cell division patterns, positional information is still the key determinant of cell fate. Received: 27 August 1999 / Accepted: 4 December 1999     

Three maize root-specific genes are not correctly expressed in regenerated caps in the absence of the quiescent center

The quiescent center is viewed as an architectural template in the root apical meristem of all angiosperm and gymnosperm root tips. In roots of Arabidopsis thaliana (L.) Heynh., the quiescent center inhibits differentiation of contacting initial cells and maintains the surrounding initial cells as stem cells. Here, the role of the quiescent center in the development of the maize (Zea mays L.) root cap has been further explored. Three maize root-specific genes were identified. Two of these were exclusively expressed in the root cap and one of them encoded a GDP-mannose-4,6-dehydratase. Most likely these two genes are structural, tissue-specific markers of the cap. The third gene, a putative glycine-rich cell wall protein, was expressed in the cap and in the root epidermis and, conceivably is a positional marker of the cap. Microsurgical and molecular data indicate that the quiescent center and cap initials may regulate the positional and structural expression of these genes in the cap and thereby control root cap development. Received: 22 September 1999 / Accepted: 9 November 1999     

Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots

Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.     

SHOOT APEX ORGANIZATION AND ORIGIN OF THE RHIZOME-BORNE ROOTS AND THEIR ASSOCIATED GAPS IN DENNSTAEDTIA CICUTARIA

The shoot apex of Dennstaedtia cicutaria consists of three zones—a zone of surface initials, a zone of subsurface initials, and a cup-shaped zone that is subdivided into a peripheral region and central region. A diffuse primary thickening meristem, which is continuous with the peripheral region of the cup-shaped zone, gives rise to a broad cortex. The roots occurring on the rhizomes are initiated very near the shoot apex in the outer derivatives of the primary thickening meristem. The roots that occur on the leaf bases also differentiate from cortical cells. Eventually, those cortical cells situated between the newly formed root apical cell and the rhizome procambium (or leaf trace) differentiate into the procambium of the root trace, thus establishing procambial continuity with that of the rhizome or leaf trace. Parenchymatous root gaps are formed in the rhizome stele and leaf traces when a few of their procambial cells located directly above the juncture of the root trace procambium differentiate into parenchyma. As the rhizome procambium or leaf trace continues to elongate, the parenchyma cells of the gap randomly divide and enlarge, thus extending the gap.     

Tornado1 and tornado2 are required for the specification of radial and circumferential pattern in the Arabidopsis root

The cell layers of the Arabidopsis primary root are arranged in a simple radial pattern. The outermost layer is the lateral root cap and lies outside the epidermis that surrounds the ground tissue. The files of epidermal and lateral root cap cells converge on a ring of initials (lateral root cap/epidermis initial) from which the epidermal and lateral root cap tissues of the seedling are derived, once root growth is initiated after germination. Each initial gives rise to a clone of epidermal cells and a clone of lateral root cap cells. These initial divisions in the epidermal/lateral root cap initial are defective in tornado1 (trn1) and trn2 plants indicating a requirement for TRN1 and TRN2 for initial cell function. Furthermore, lateral root cap cells develop in the epidermal position in trn1 and trn2 roots indicating that TRN1 and TRN2 are required for the maintenance of the radial pattern of cell specification in the root. The death of these ectopic lateral root cap cells in the elongation zone (where lateral root cap cells normally die) results in the development of gaps in the epidermis. These observations indicate that TRN1 and TRN2 are required to maintain the distinction between the lateral root cap and epidermis and suggest that lateral root cap fate is the default state. It also suggests that TRN1 and TRN2 repress lateral root cap fate in cells in the epidermal location. Furthermore, the position-dependent pattern of root hair and non-root hair cell differentiation in the epidermis is defective in trn1 and trn2 mutants. Together these results indicate that TRN1 and TRN2 are required for the maintenance of both the radial pattern of tissue differentiation in the root and for the subsequent circumferential pattern within the epidermis.     

Constitutive autophagy in plant root cells

In previous studies, using a membrane-permeable protease inhibitor, E-64d, we showed that autophagy occurs constitutively in the root cells of barley and Arabidopsis. In the present study, a fusion protein composed of the autophagy-related protein AtAtg8 and green fluorescent protein (GFP) was expressed in Arabidopsis to visualize autophagosomes. We first confirmed the presence of autophagosomes with GFP fluorescence in the root cells of seedlings grown on a nutrient-sufficient medium. The number of autophagosomes changed as the root cells grew and differentiated. In cells near the apical meristem, autophagosomes were scarcely found. However, a small but significant number of autophagosomes existed in the elongation zone. More autophagosomes were found in the differentiation zone where cell growth ceases but the cells start to form root hair. In addition, we confirmed that autophagy is activated under starvation conditions in Arabidopsis root cells. When the root tips were cultured in a sucrose-free medium, the number of autophagosomes increased in the elongation and differentiation zones, and a significant number of autophagosomes appeared in cells near the apical meristem. The results suggest that autophagy in plant root cells is involved not only in nutrient recycling under nutrient-limiting conditions but also in cell growth and root hair formation.     

ABA promotes quiescence of the quiescent centre and suppresses stem cell differentiation in the Arabidopsis primary root meristem

It is well known that abscisic acid (ABA) can halt meristems for long periods without loss of meristem function, and can also promote root growth at low concentrations, but the mechanisms underlying such regulation are largely unknown. Here we show that ABA promotes stem cell maintenance in Arabidopsis root meristems by both promoting the quiescence of the quiescent centre (QC) and suppressing the differentiation of stem cells and their daughters. We demonstrate that these two mechanisms of regulation by ABA involve distinct pathways, and identify components in each pathway. Our findings demonstrate a cellular mechanism for a positive role for ABA in promoting root meristem maintenance and root growth in Arabidopsis.