Diversity of cell lengths in terminal portions of roots: implications to cell proliferation

Terminal meristems are responsible for all primary growth of roots. It has been asserted that all cells of root meristems are actively dividing (no cells cycle slowly or arrest in the cycle) and stem cell populations expand exponentially. Because cells do not slide relative to each other in roots, relative cell lengths may be used to determine relative cell cycle durations and/or proportions of cells actively dividing in root tissues. If all cells are cycling, no interphase cells should be longer than critical length (length of longest mitotic cell in the meristem) and cells should exhibit an exponential cell-age distribution. Lengths of all cells were obtained radially across entire median longitudinal root sections at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mm from the founder cell/root cap boundary for five plant species to estimate percentages of cells longer than critical length. For example, up to 15 and 90% of all interphase cells were longer than critical length in 0.5 and 2.0 mm tissues, respectively, indicating that slow-cycling and/or non-proliferative cells are present in such tissues. In order to determine if the distribution of cell lengths in 0.5 mm segments approximated an exponential cell-age distribution, lengths of interphase cells less than critical length were determined. Such interphase cells were placed into ten groups according to cell length and percentages of cells in each group were compared with percentages of cells in groups calculated from an exponential cell-age distribution. Percentages of cells were significantly different from predicted percentages of between 6 and 9 out of ten groups - cell lengths were not distributed exponentially. Because there are significant numbers of interphase cells longer than critical length and since lengths of interphase cells shorter than critical length do not resemble an exponential cell-age distribution, it must be concluded that not all cells in root segments from 0.5 to 3.0 mm root segments are actively dividing. Heretofore, no databases of cell lengths have been used to test these assertions.     

3D analysis of mitosis distribution highlights the longitudinal zonation and diarch symmetry in proliferation activity of the Arabidopsis thaliana root meristem

To date CYCB1;1 marker and cortex cell lengths have been conventionally used to determine the proliferation activity of the Arabidopsis root meristem. By creating a 3D map of mitosis distribution we showed that these markers overlooked that stele and endodermis save their potency to divide longer than the cortex and epidermis. Cessation of cell divisions is not a random process, so that mitotic activity within the endodermis and stele shows a diarch pattern. Mitotic activity of all root tissues peaked at the same distance from the quiescent center (QC); however, different tissues stopped dividing at different distances, with cells of the protophloem exiting the cell cycle first and the procambial cells being the last. The robust profile of mitotic activity in the root tip defines the longitudinal zonation in the meristem with the proliferation domain, where all cells are able to divide; and the transition domain, where the cell files cease to divide. 3D analysis of cytokinin deficient and cytokinin signaling mutants showed that their proliferation domain is similar to that of the wild type, but the transition domain is much longer. Our data suggest a strong inhibitory effect of cytokinin on anticlinal cell divisions in the stele.     

Analysis of cell cycle in root meristems

We have analyzed the cell proliferation in a meristem assuming a single file model for root architecture. The meristem file appears to be built up by two clearly separated zones: the first going from the initial cell to the middle of the meristem and the second from the middle to the meristem boundary. The first half of the meristem shows an exponential age distribution for the cell population. In contrast, in the second half of the meristem, the cell kinetics of cycling cells strongly disagree with exponential kinetics and due to the compensation between the observed deviations in both halves, cell supply in the file meristem is in line with linear kinetics. However, we proposed that exponential kinetic equations offer a suitable approach to problems of cell cycle compartments and population age distributions in real meristems, where non-cycling cells cannot be identified inside the meristem, whether we consider the meristem as a whole or study a “window” inside it. Nevertheless, for more exact kinetic analysis, when estimating the proliferative fraction, the width of the “window” and its location along the axis must carefully be taken into account.     

Variations in the size of mitotic cells in the root meristem

Variations in the length of mitotic and interphase cells were analyzed in various tissues of wheat roots and in the cortex of maize roots. Reliable differences were shown in the length of mitotic cells in individual files-clones of cells of the same tissue. The mean lengths of dividing cells in different roots differed to a lesser extent than those of different files in the same tissue of one root. Within the file, the length of sister simultaneously dividing cells differed the least, while the difference of lengths of neighbor simultaneously dividing nonsister cells was bigger. The mean length of interphase cells in any file was always less than that of mitotic cells by a factor of 1.45. This ratio was almost invariable for files and tissues in both plants we studied and corresponded to that of an exponentially growing cell population. In addition, a very small number of cells were found (less than 1%) in meristems, which are longer than the mitotic cells. The length of these cells exceeded those of mitotic cells by less than twice. The origin of such cells is discussed. The length of mitotic cells near the quiescent center is more variable than in the middle of the meristem in the cortex of both plants. In the meristem basal part, the mitotic cells were no longer than those in the middle of the meristem but there were no small dividing cells. In the wheat epidermis, the cells are differentiated into trichoblasts and atrichoblasts and, therefore, the length of dividing cells is highly variable. The cell length is essential for their transition to mitosis for all studied proliferating meristem cells.     

Variations in the Size of Mitotic Cells in the Root Meristem

Variations in the length of mitotic and interphase cells were analyzed in various tissues of wheat roots and in the cortex of maize roots. Reliable differences were shown in the length of mitotic cells in individual file clones of cells of the same tissue. The mean lengths of dividing cells in different roots differed to a lesser extent than those of different files in the same tissue of one root. Within the file, the length of the sister simultaneously dividing cells differed the least, while the difference of lengths of the neighbor simultaneously dividing nonsister cells was bigger. The mean length of interphase cells in any file was always less than that of mitotic cells by a factor of 1.45. This ratio was almost invariable for files and tissues in both the plants we studied and corresponded to that of an exponentially growing cell population. In addition, a very small number of cells were found (less than 1%) in meristems, which are longer than the mitotic cells. The length of these cells exceeded those of mitotic cells by less than twice. The origin of such cells is discussed. The length of mitotic cells near the quiescent center is more variable than in the middle of the meristem in the cortex of both plants. In the meristem basal part, the mitotic cells were no longer than those in the middle of the meristem but there were no small dividing cells. In the wheat epidermis, the cells are differentiated into trichoblasts and atrichoblasts and, therefore, the length of the dividing cells is highly variable. The cell length is essential for their transition to mitosis for all studied proliferating meristem cells.     

Characteristics of root apical meristem cell population kinetics: A review of analyses and concepts

Various conclusions have been reached in the literature concerning the organization of root meristems in terms of cell proliferation and quiescence. In this article the methods used in such analyses are examined in order to assess the validity of the results obtained. Cells in root meristems are clearly heterogeneous in terms of cycle time. The existence of quiescent populations of cells outside the quiescent center, however, is more problematic because (i) it is difficult to define clearly the proximal boundary of the root apical meristem and (ii) present techniques used to measure the size of the proliferative cell fraction are likely to be, in many, if not all cases, highly inaccurate. We have concluded that non-cycling cells, other than those of the quiescent center, do not exist within the meristem proper at the apex of the proliferating angiosperm root.     

The Difference Between Open and Closed Meristems

An open and a closed root meristem have been compared by investigatingthe cell kinetics of small regions of the apices of Helianthusand Zea. The cells of the stelar pole are quiescent in both and thereis no exchange of cells between stele and cortex or stele andcap. The immediately distal cells in the closed meristem (Zea)are also quiescent and the few divisions that do occur can betransverse or longitudinal. In the open meristem (Helianthus)these cells are not quiescent, but they go out of cycle transiently,prolonging the potential cell-doubling time. Their divisionsare transverse. It is a consequence of these differences thatclosed meristems form root caps discrete from the cortex whereasopen meristems force instability in the boundary between theperipheral part of the cap and the cortex. Another consequencein roots with open meristems is a succession of columella complexestransversely displaced from each other by the state of fluxin the meristem during the non-cycling phase of the proximaltier of cells, those immediately distal to the stelar pole. The results are discussed in relation to the ontogenetic onsetof quiescence and the evidence for switches between open andclosed operation of meristems. meristem, root apex, Helianthus annuus, Zea mays L.     

The age-distribution of cell cycle populations in plant root meristems : Complex tissues

The durations of the mitotic cycle periods, the proportions of cells in each cycle period of the proliferative and nonproliferative populations, and the rates of cell progression from G1-S and from G2-M were used to characterize the cytokinetics in root meristems of four plant species. The observed age-distribution of cells in the cycle of each meristem was not comparable to either a theoretical exponential or uniform age-distribution. A more exact fit of the observed age-distributions with theoretical distributions was hindered by proliferative cells which halted temporarily in G1 and/or in G2 in relative proportions similar to the nonproliferative cells in the meristem. Moreover, the preponderant cycle period in which cells halted temporarily differed among the species observed: it was G1 in Helianthus and Triticum and G2 in Pisum and Vicia. The cell populations of these complex tissues can be subdivided kinetically into three types: (1) rapidly proliferating cells; (2) slowly proliferating cells that halt temporarily in G1 and/or in G2; and (3) nonproliferating cells that halt in G1 and/or in G2.     

Cellular parameters of the shoot apical meristem in Arabidopsis.

The shoot apical meristem (SAM) is a small group of dividing cells that generate all of the aerial parts of the plant. With the goal of providing a framework for the analysis of Arabidopsis meristems at the cellular level, we performed a detailed morphometric study of actively growing inflorescence apices of the Landsberg erecta and Wassilewskija ecotypes. For this purpose, cell size, spatial distribution of mitotic cells, and the mitotic index were determined in a series of optical sections made with a confocal laser scanning microscope. The results allowed us to identify zones within the inflorescence SAM with different cell proliferation rates. In particular, we were able to define a central area that was four to six cells wide and had a low mitotic index. We used this technique to compare the meristem of the wild type with the enlarged meristems of two mutants, clavata3-1 (clv3-1) and mgoun2 (mgo2). One of the proposed functions of the CLV genes is to limit cell division rates in the center of the meristem. Our data allowed us to reject this hypothesis, because the mitotic index was reduced in the inflorescence meristem of the clv3-1 mutant. We also observed a large zone of slowly dividing cells in meristems of clv3-1 seedlings. This zone was not detectable in the wild type. These results suggest that the central area is increased in size in the mutant meristem, which is in line with the hypothesis that the CLV3 gene is necessary for the transition of cells from the central to the peripheral zone. Genetic and microscopic analyses suggest that mgo2 is impaired in the production of primordia, and we previously proposed that the increased size of the mgo2 meristem could be due to an accumulation of cells at the periphery. Our morphometric analysis showed that mgo2 meristems, in contrast to those of clv3-1, have an enlarged periphery with high cell proliferation rates. This confirms that clv3-1 and mgo2 lead to meristem overgrowth by affecting different aspects of meristem function.     

狭叶柴胡根的发育解剖学研究

应用常规石蜡切片法对狭叶柴胡（Bupleurum scorzonerifolium Willd.）根的发育过程进行了解剖学研究,并对其1年生与多年生根的结构进行了比较。结果表明,狭叶柴胡根的发育包括原分生组织、初生分生组织、初生结构和次生生长4个发育阶段。原分生组织由3群原始细胞组成,其细胞具有典型分生组织的细胞学特征;初生分生组织包括根冠原、表皮原、皮层原和中柱原。初生结构由表皮、皮层和中柱组成。初生木质部多为二原型,少数为三原型。次生结构为：从外到内由周皮、中柱鞘薄壁细胞环和次生维管组织组成,次生生长主要是依靠维管形成层和木栓形成层的活动来完成,其木栓形成层由中柱鞘细胞恢复分裂能力而形成。多年生根与一年生根的结构基本相似,但在各部分的细胞数量和组成上存在差异。分泌道在一年生的根中仅分布在中柱鞘薄壁组织中,而在多年生的根中,在中柱鞘薄壁细胞和次生韧皮部中均有分布。     

Embryo-patterning genes and reinforcement cues determine cell fate in theArabidopsis thalianaroot

The majority of plant organs arise from groups of continuously dividing cells, the meristems. Little is known about mechanisms of cell specification in meristems. Within theArabidopsisroot meristem, the fate of every cell can be predicted accurately, and the origin of these cells during the formation of the embryonic root primordium is known. Laser ablations reveal that, despite the regularity in cell lineage, position remains important to reinforce cell specification. Genetic analysis has revealed that many genes involved in the specification of the main cell types in the root act early, during embryogenesis, and an important question is whether the same or other genes are involved in the reinforcement of specification. Sub-specification of cell types, as exemplified by epidermal root hair cell specification, involves two pathways, one of which may act to reinforce earlier patterning events mediated by the other.     

Meristem as a Self-Renewing System: Maintenance and Cessation of Cell Proliferation (A Review)

The mechanisms of the maintenance of long-term cell proliferation and its cessation in the meristem of the growing root were analyzed. Quiescent center (QC) remains in the meristem for a long time, whereas all other cells leave the meristem after several mitotic cycles. The question arises as to what extent such organization of proliferation corresponds to the concept of stem cells elaborated for animals. The definition of animal stem cells is met by the QC cells rather than by actively dividing cells that boundary it. However, QC is not a self-maintaining population of cells originated during early stages of embryogenesis; it is formed from dividing cells in the main or lateral root. After root decapitation, the QC can arise from the cells that normally would leave the meristem before long. There is a zone of the meristem whose cells are capable of remaining and forming QC after the removal of the apical part of the root. Maintenance of the size of the meristem depends on the interaction between QC, initial cells located at its surface, and the actively dividing cells. Apparently, the life span of cells in the meristem determines the time when the meristematic cell will begin the elongation. The number of cells starting the elongation depends on proliferation rate and on the changes in life span of meristematic cells which determine their initial number. The life span of the cells in the meristem for most actively dividing cells does not depend on the cell divisions, and remains unchanged in the presence of various inhibitors. As a result of inhibited proliferation in the main part of the meristem, cell divisions in the QC are activated and newly formed cells may proceed to rapid divisions. Thus, the size of the meristem is maintained by the operation of several mechanisms, and individual processes may be, on the one hand, relatively independent and, on the other hand, regulated either by feedback or directly. As a result, the root growth becomes resistant to various external events.     

An Early Cytochemical Marker of Commitment to Stelar Differentiation in Meristems from Dicotyledonous Plants

A cytochemical study of root apices from Vicia faba and Pisumsativum showed esterase activity to be present in the stele,root cap and rhizodermis, but almost completely absent fromthe developing cortex and quiescent centres. The meristem cellsgiving rise to the cortex were almost negative whilst thosegiving rise to the stele were positive for esterase activity.Cambia from roots, shoots and petioles of a number of dicotyledonousspecies were all positive for esterase activity. It is proposedthat esterase activity may be used as an early marker of commitmentto differentiation into stele in roots of dicotyledonous plants,and that the cambia are fully committed meristems. Pisum sativum L., Vicia fabaL., garden pea, broad bean, meristems, stelar differentiation, esterase activity, xylem differentiation, cytochemistry, cambium     

The extent and differences in mitotic activity of the root tip ofVicia faba L.

Mitotic activity does not stop for different meristematic cells of the root apex at the same distance from the initials. The differences are connected with the functional heterogeneity of the apical meristem of the root. The arrangement of vascular bundles,i.e. the alternation of independent xylem and phloem groups, is of major importance. In broad bean roots, the protophloem sieve elements stop dividing first. The centre of the stelei. e. late metaxylem elements stop dividing next. Division in the stele gradually ceases centrifugally, while it ceases centripetally in the peripheral part of the root. The cylindrical region with prolonged cell division includes internal layers of the cortex including endodermis, pericycle and adjoining cells of the stele. Proximally apical meristem is reduced to isolated strands of cells adjacent to the protoxylem poles. Pericycle cells stop dividing last at a distance of approx. 9–10 mm from the initials. The number of the division cycles is limited and is specific for individual cell types. Epidermal and cortical cells divide in broad bean roots transversely approximately seven times, cells of late metaxylem approximately five times. Root apical meristem is an asynchronous cell population with a different duration of the mitotic cycle. We determined local variations in the duration of the mitotic cycle in the apical meristem of broad bean root by means of colchicine-induced polyploidy. The cells of the quiescent centre had the longest mitotic cycle after colchicine treatment. The region of the proper root adjacent to the quiescent centre was mixoploid (2n and 4n). Isolated cells with a long cycle occurred also in the cortex and in the central cylinder. Cells with a division cycle of 18h were found in the root cap, in the epidermis, in the cortex and in the central cylinder. Relatively numerous cells with the shortest division cycle, approx. 12 h, occurred farther of the quiescent centre in the epidermis, in the cortex, in the pericycle, and in adjacent layers of the stele through-out the entire meristematic region. The results derived from the analysis of the apical meristem are discussed in connection with the ontogenesis of different types of cells taking part in the primary structure of the root.     

Exponential pattern of cell age distribution in dividing cells of plant meristems

In order to determine the pattern of cell age distribution in proliferating cells of Allium cepa roots we have measured by cytophotometry two cell size parameters, protein content and surface area projection, in cells that correspond to the entire proliferating population or only to the ana-telophase subpopulation. The size values of ana-telophase cells have been employed to construct theoretical size distributions for the entire proliferating cell population of the root meristem by assuming either a uniform or an exponential cell age distribution. Statistical comparison of theoretical distributions with the experimental one rules out a uniform cell age distribution and strongly favours an exponential age distribution similar to that found in bacteria.     

Mutations in the Diageotropica (Dgt) gene uncouple patterned cell division during lateral root initiation from proliferative cell division in the pericycle

In angiosperms, root branching requires a continuous re-initiation of new root meristems. Through some unknown mechanism, in most eudicots pericycle cells positioned against the protoxylem change identity and initiate patterned division, leading to formation of lateral root primordia that further develop into lateral roots. This process is auxin-regulated. We have observed that three mutations in the Diageotropica (Dgt) gene in tomato prevent primordium formation. Detailed analysis of one of these mutants, dgt1-1, demonstrated that the mutation does not abolish the proliferative capacity of the xylem-adjacent pericycle in the differentiated root portion. Files of shortened pericycle cells found in dgt1-1 roots were unrelated to primordium formation. Auxin application stimulated this unusual proliferation, leading to formation of a multi-layered xylem-adjacent pericycle, but did not rescue the primordium formation. In contrast to wild type, auxin could not induce any cell divisions in the pericycle of the most distal dgt1-1 root-tip portion. In wild-type roots, the Dgt gene promoter was expressed strongly in lateral root primordia starting from their initiation, and on auxin treatment was induced in the primary root meristem. Auxin level and distribution were altered in dgt1-1 root tissues, as judged by direct auxin measurements, and the tissue-specific expression of an auxin-response reporter was altered in transgenic plants. Together, our data demonstrate that the Dgt gene product, a type-A cyclophilin, is essential for morphogenesis of lateral root primordia, and that the dgt mutations uncouple patterned cell division in lateral root initiation from proliferative cell division in the pericycle.     

THE OCCURRENCE OF INTERCALARY AND UNINTERRUPTED MERISTEMS IN THE INTERNODES OF TROPICAL MONOCOTYLEDONS

The distribution of percent of dividing nuclei, parenchyma cell length, total cell number per internode, and total internode length were determined for successive internodes in the apex and growing vegetative internodes of 23 tropical species in 17 families of monocotyledons. Basal intercalary meristems (IM) were found in representatives of Commelinaceae, Cyperaceae, Flagellariaceae, Poaceae, Restionaceae, and Marantaceae. Uninterrupted meristems (UM) which are confined progressively to the upper region of the internode and are not isolated meristematic regions were found in the Costaceae, Dioscoreaceae, Philesiaceae, Smilacaceae, Agavaceae, Araceae, Arecaceae, Liliaceae, Pandanaceae, and Zingiberaceae. Both IM and UM were found in different species of Orchidaceae. The only morphological trait correlated with meristem type was presence of sheathing leaf bases in all species with IM. Both IM and UM are interpreted as extensions of the primary elongating meristem; the IM is disjunct, and the UM is continuous with it. The phytomer growth unit and the presence of internodal IM's cannot be applied generally to the monocotyledons.     

On the constancy of cell division rate in the root meristem

This review examines under what circumstances the rate of cell division among cells of the root meristem is known to vary. First, methods are compared that have been used to quantify cell division rate. These can be grouped as being either cytological, in which the rate of accumulation of cells in a particular phase of the cell cycle is determined based on some form of cytological labeling, or kinematic, in which the rate of cell accumulation is determined from the net movement of cells. Then, evidence is reviewed as to whether cell division rates vary between different tissues or cell types, between different positions in the root, or finally between different environments. The evidence is consistent with cells dividing at a constant rate, and well documented examples where cell division rate changes substantially are rare. The constancy of cell division rate contrasts with the number of dividing cells, which varies extensively, and implies that a major point for cell cycle control is governing the exit from the proliferative state at the basal boundary of the meristem.     

Comparative anatomy of embryogenesis in three species of Podostemaceae and evolution of the loss of embryonic shoot and root meristems

During embryogenesis in angiosperms, the embryonic shoot and root meristems are created at opposite poles of the embryo, establishing a vertical body plan. However, the aquatic eudicot family Podostemaceae exhibits an unusual horizontal body plan, which is attributed to the loss of embryonic shoot and root meristems. To infer the embryogenetic changes responsible for the loss of these meristems, we examined the embryogenesis of three podostemads with different meristem characters, that is, Terniopsis brevis with distinct shoot and root meristems, Zeylanidium lichenoides with reduced shoot and no root meristems, and Hydrobryum japonicum with no shoot and no root meristems. In T. brevis, as in other eudicots, the putative organizing center (OC) and L1 layer (=the epidermal cell layer) arose to generate a distinct shoot meristem initial, and the hypophysis formed the putative quiescent center (QC) of a root meristem. Z. lichenoides had a morphologically unrecognizable shoot meristem, because a distinct L1 layer did not develop, whereas the putative OC precursor arose normally. In H. japonicum, the vertical divisions of the apical cells of eight-cell embryo prevented putative OC initiation. In Z. lichenoides and H. japonicum, the putative QC failed to initiate because the hypophysis repeated longitudinal divisions during early embryogenesis. Based on their phylogenetic relationships, we infer that the conventional embryonic shoot meristem was lost in Podostemaceae via two steps, that is, the loss of a distinct L1 layer and the loss of the OC, whereas the loss of the embryonic root meristem occurred once by misspecification of the hypophysis.     

In vivo analysis of cell division, cell growth, and differentiation at the shoot apical meristem in Arabidopsis

The aerial parts of the plant are generated by groups of rapidly dividing cells called shoot apical meristems. To analyze cell behavior in these structures, we developed a technique to visualize living shoot apical meristems using the confocal microscope. This method, combined with green fluorescent protein marker lines and vital stains, allows us to follow the dynamics of cell proliferation, cell expansion, and cell differentiation at the shoot apex. Using this approach, the effects of several mitotic drugs on meristem development were studied. Oryzalin (depolymerizing microtubules) very rapidly caused cell division arrest. Nevertheless, both cell expansion and cell differentiation proceeded in the treated meristems. Interestingly, DNA synthesis was not blocked, and the meristematic cells went through several rounds of endoreduplication in the presence of the drug. We next treated the meristems with two inhibitors of DNA synthesis, aphidicolin and hydroxyurea. In this case, cell growth and, later, cell differentiation were inhibited, suggesting an important role for DNA synthesis in growth and patterning.