Lateral root initiation by asymmetrical transverse divisions of pericycle cells in adventitious roots ofAllium cepa

Summary In onion adventitious roots cellular events have been identified that indicate that lateral root initiation occurs earlier and nearer the apex than previously documented. Lateral roots are not initiated when a pericycle cell divides periclinally but earlier, when a pair of neighbouring pericycle cells in the same column divide transversely and asymmetrically, with both mitoses close to the end towards the neighbouring pericycle cell. Each cell therefore produces two cells of unequal length. The shorter cells produced by the mother pericycle cells are adjacent, while the longer cells are located above and below the shorter cells. This objective morphological criterion allows clear identification of the site of lateral root initiation. Subsequent to these asymmetric divisions, both the longer pericycle cells again divide transversely and asymmetrically producing more short cells adjacent to the previous ones. The first periclinal division occurs in one of these short pericycle cells.     

Pericycle proliferation pattern during the lateral root initiation in adventitious roots ofAllium cepa

Summary Far from the apical meristem of adventitious roots ofAllium cepa, the pericycle shows great proliferative activity related to lateral root initiation. A group of mother pericycle cells undergoes asymmetrical transverse and periclinal divisions following a well-established pattern. Successive asymmetrical transverse divisions, progressing from one end of the cell to the other, divide the original mother cell into very short derivatives. Later, these short derivative cells undergo periclinal divisions. This proliferative activity starts nearly simultaneously in two elongated and highly vacuolated pericycle cells located in the same column in front of one of the xylem poles. Then proliferation expands centrifugally towards other pericycle cells in the same and adjacent columns. The proliferative activity of the pericycle cells decreases progressively outwards. Only the most central of these cells produce derivatives which contribute to the future lateral root.     

DEVELOPMENTAL ANATOMY IN ROOTS OF IPOMOEA PURPUREA. II. INITIATION AND DEVELOPMENT OF SECONDARY ROOTS

In seedlings of Ipomoea purpurea secondary roots are initiated in the primary root pericycle opposite immature protoxylem. Cells derived from immature endodermis, pericycle, and incipient protoxylem and stelar parenchyma contribute to the primordium. The derivatives of the endodermis become a uniseriate covering over the tip and flanks of the primordium and emerged secondary root; the endodermal covering is sloughed off when the lateral root reaches 1–5 mm in length. A series of periclinal and anticlinal divisions in the pericycle and its derivatives gives rise to the main body of the secondary root. The initials for the vascular cylinder, cortex, and rootcap-epidermis complex are established very early during primordium enlargement. After emergence from the primary root, the cortical initials undergo significant structural modifications related to enlargement of the ground meristem and cortex, and the rootcapepidermal initials are partitioned into columellar initials and lateral rootcapepidermal initials. Procambium diameter increases by periclinal divisions in peripheral sectors. The mature vascular cylinder is comprised of several vascular patterns, ranging from diarch to pentarch, that are probably related ontogenetically. Cells derived from incipient protoxylem and stelar parenchyma cells of the primary root form the vascuar connection between primary and secondary roots.     

Pericycle cell proliferation and lateral root initiation in Arabidopsis

In contrast with other cells generated by the root apical meristem in Arabidopsis, pericycle cells adjacent to the protoxylem poles of the vascular cylinder continue to cycle without interruption during passage through the elongation and differentiation zones. However, only some of the dividing pericycle cells are committed to the asymmetric, formative divisions that give rise to lateral root primordia (LRPs). This was demonstrated by direct observation and mapping of mitotic figures, cell-length measurements, and the histochemical analysis of a cyclin-GUS fusion protein in pericycle cells. The estimated duration of a pericycle cell cycle in the root apical meristem was similar to the interval between cell displacement from the meristem and the initiation of LRP formation. Developmentally controlled LRP initiation occurs early, 3 to 8 mm from the root tip. Thus the first growth control point in lateral root formation is defined by the initiation of primordia in stochastic patterns by cells passing through the elongation and young differentiation zones, up to where lateral roots begin to emerge from the primary root. Therefore, the first growth control point is not restricted to a narrow developmental window. We propose that late LRP initiation is developmentally unrelated to the root apical meristem and is operated by a second growth control point that can be activated by environmental cues. The observation that pericycle cells divide and lateral root primordia form without intervening mitotic quiescence suggests that lateral organ formation in roots and shoots might not be as fundamentally different as previously thought.     

A histological study of lateral root initiation and development inZea mays

Summary A light microscopic study has been made of the origin and development of lateral roots inZea mays.The initiation of a lateral occurs adjacent to a xylem pole and involves an increase in cytoplasmic basophilia and subsequent divisions of cells of the pericycle and the parenchyma of the stele of the mother root.Cells derived from the parent pericycle form most of the young lateral but its epidermis and root cap are composed of cells of endodermal origin.Two different types of polysaccharides are secreted by cells of the young lateral root. One type which is PAS-positive and non-metachromatic, is produced by the epidermal cells, while the other type, metachromatic and only slightly PAS-positive, is secreted by the root cap cells.     

Resumption of DNA Synthesis and Cell Division in Wheat Roots as Related to Lateral Root Initiation

The arrest of DNA synthesis and termination of cell division in basal meristematic cells as well as the resumption of these processes as related to the initiation of lateral root primordia (LRP) were studied in tissues of Triticum aestivumroots incubated with ³H-thymidine. All cells of the stelar parenchyma and cortex as well as most endodermal and pericycle cells left the mitotic cycle and ceased proliferative activity at the basal end of the meristem and at the beginning of the elongation zone. Some endodermal and pericycle cells started DNA synthesis in the basal part of the meristem and completed it later on during their elongation, but they did not divide. In the cells of these tissues, DNA synthesis resumed above the elongation zone, the cells being located much closer to the root tip than the first newly dividing cells. Thus, the initiation of LRP started much closer to the root tip than it was previously believed judging from the distance of the first dividing pericycle cells from the root tip. DNA synthesizing and dividing cells first appeared in the stelar parenchyma, then, in the pericycle, and later, in the endodermis and cortex. It seems likely that a release from the inhibition of DNA synthesis allows the cells that completed mitotic cycle in the basal part of meristem in the G₁phase to cease the proliferative arrest above the elongation zone and to continue their cycling. The location of the first DNA synthesizing and dividing cells in the stelar parenchyma and pericycle did not strictly correspond to the LRP initiation sites and proximity to the xylem or phloem poles. This indicates that LRP initiation results from the resumption of DNA synthesis in all pericycle and stelar parenchyma cells that retained the ability to synthesize DNA and occurs only in the pericycle sector situated between the two tracheal protoxylem strands, all cells of which terminated their mitotic cycles in the G₁phase.     

GROWTH AND DEVELOPMENT OF CULTURED RADISH ROOTS

Excised cultured roots of Raphanus sativus L. cv. White Icicle elongate and produce a few lateral roots but do not increase in diameter. Lateral expansion is effected when both an auxin (indoleacetic acid or naphthaleneacetic acid) and a cytokinin (benzyladenine) are applied at the cut end of the root. The growth regulator effects are apparent first in the pericycle and subsequently in the procambium. Both of these groups of cells divide, producing large numbers of secondary derivatives. The increase in number of cells is reflected in an increase in root diameter. When cultured roots are treated with auxin only, a limited number of pericycle cells divide and lateral roots develop. When roots are treated with cytokinin only, all pericycle cells divide and a multiseriate zone of pericycle-derived cells develops. The procambium is not markedly affected by application of a single growth regulator. The distinct and separable responses of pericycle cells to different regulators suggest that the pericycle can be characterized in a functional, as well as a topographic, sense.     

Lateral Root Initiation or the Birth of a New Meristem

Root branching happens through the formation of new meristems out of a limited number of pericycle cells inside the parent root. As opposed to shoot branching, the study of lateral root formation has been complicated due to its internal nature, and a lot of questions remain unanswered. However, due to the availability of new molecular tools and more complete genomic data in the model species Arabidopsis, the probability to find new and crucial elements in the lateral root formation pathway has increased. Increasingly more data are supporting the idea that lateral root founder cells become specified in young root parts before differentiation is accomplished. Next, pericycle founder cells undergo anticlinal asymmetric, divisions followed by an organized cell division pattern resulting in the formation of a new organ. The whole process of cell cycle progression and stimulation of the molecular pathway towards lateral root initiation is triggered by the plant hormone auxin. In this review, we aim to give an overview on the developmental events taking place from the very early specification of founder cells in the pericycle until the first anticlinal divisions by combining the knowledge originating from classical physiology studies with new insights from genetic-molecular analyses. Based on the current knowledge derived from recent genetic and developmental studies, we propose here a hypothetical model for LRI.     

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.     

Cytokinins act directly on lateral root founder cells to inhibit root initiation

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.     

EXPERIMENTAL STUDIES OF HAUSTORIUM INITIATION AND EARLY DEVELOPMENT IN AGALINIS PURPUREA (L.) RAF. (SCROPHULARIACEAE)

In parasitic angiosperms the haustorium, an organ specialized for attachment and penetration of host tissue, functions in the transport of water and nutrients from the host to the parasite. In Agalinis purpurea (L.) Raf. (Scrophulariaceae) these organs are initiated laterally along its roots, opposite a primary xylem pole. Analyses of haustoria distribution and cellular root profiles show that the portion of the root which is most sensitive to haustorial elicitor molecules is the area distal to the zone of elongation and near the root meristem. Sectioned material supports this finding and, further, indicates that the cells which are the first to respond to haustorial elicitors are located in the inner cortex. Haustoria develop rapidly in response to a host root or to isolated chemical elicitors (xenognosins) normally contained in host root exudate. By 6 hr, vacuolation and radial cellular enlargement are observed in the cortex, and a lateral swelling along the root is visible. By 12 hr, cells of the epidermis divide anticlinally to establish a group of densely cytoplasmic cells at the apex of the haustorial swelling. Accompanying these divisions is the differentiation of specialized hair cells which elongate from epidermal cells flanking the presumptive haustorial apex. Next, the internal, radially enlarged cortical cells divide periclinally. Periclinal divisions are subsequently initiated in the pericycle as early as 18 hr post-induction. Cellular division and enlargement continue so that by 24–36 hr a mature pre-contact haustorium is formed. There is a reduction in root elongation concomitant with haustorial initiation. Depending upon the number of haustoria produced, elongation typically returns to the preinduction level within 2 or 3 days.     

Origin of Epidermis and Development of Root Primordia in Pistia, Hydrocharis and Eichhornia

All three floating plants have roots bearing laterals derivedfrom both pericycle and endodermis. In Pistia and Eichhornialaterals arise within the meristem of the mother root; in Hydrocharisthey arise from mature tissue. In Pistia and Hydrocharis theepidermis becomes anatomically discrete between cortex and cap:in Pistia it is derived from the endodermis of the mother root,in Hydrocharis from the pericycle. The epidermis is not discretein Eichhornia and is derived from the pericycle of the motherroot with the cortex. Stathmokinetic data were used to construct timetables of developmentwhich show how the differences arise. In Pistia the first periclinaldivision of the endodermis-derived tissue individualizes theepidermis and occurs early, before a quiescent centre forms.In Hydrocharis the epidermis also becomes discrete before thepole of the meristem becomes quiescent, but it does so by apericlinal division of the pericycle-derived tissue. In Eichhorniapericlinal divisions occur in the outermost layer of the pericycle-derivedtissue long after quiescence has set in at the pole and afterthe fourth periclinal division in the endodermis derived cap.Its epidermis therefore never becomes anatomically discretethough it becomes functionally discrete because its polar cellsstop dividing as in the other plants. The involvement of the endodermis of mother roots in the formationof laterals is discussed in relation to the state of differentiationat sites of primordium formation, discreteness of the epidermisand subsequent fate of primordia. Pistia stratiotes L., Hydrocharis morsus-ranae L., Eichhornia crassipes Solms., primordia, lateral root, discrete epidermis, development, chimera, stathmokinetics     

Phloem-associated auxin response maxima determine radial positioning of lateral roots in maize

In Arabidopsis thaliana, lateral-root-forming competence of pericycle cells is associated with their position at the xylem poles and depends on the establishment of protoxylem-localized auxin response maxima. In maize, our histological analyses revealed an interruption of the pericycle at the xylem poles, and confirmed the earlier reported proto-phloem-specific lateral root initiation. Phloem-pole pericycle cells were larger and had thinner cell walls compared with the other pericycle cells, highlighting the heterogeneous character of the maize root pericycle. A maize DR5::RFP marker line demonstrated the presence of auxin response maxima in differentiating xylem cells at the root tip and in cells surrounding the proto-phloem vessels. Chemical inhibition of auxin transport indicated that the establishment of the phloem-localized auxin response maxima is crucial for lateral root formation in maize, because in their absence, random divisions of pericycle and endodermis cells occurred, not resulting in organogenesis. These data hint at an evolutionarily conserved mechanism, in which the establishment of vascular auxin response maxima is required to trigger cells in the flanking outer tissue layer for lateral root initiation. It further indicates that lateral root initiation is not dependent on cellular specification or differentiation of the type of vascular tissue.     

Cell-cycle regulation of hydroxyproline-rich glycoprotein HRGPnt3 gene expression during the initiation of lateral root meristems

Analysis of transgenic tobacco plants containing a tobacco hydroxyproline-rich glycoprotein HRGPnt3 gene promoter-β-glucuronidase (GUS) gene fusion (HRGPnt3-uidA) showed that this promoter is active not only in the early stages of initiation of lateral roots as previously described, but also in the initiation of adventitious roots, with similar selective expression in a subset of pericycle cells. HRGPnt3 is also induced during initiation of hairy roots following transformation by Agrobacterium rhizogenes. The auxin indole acetic acid (IAA) induces an increase in the number of characteristic discrete sites of HRGP-nt3 expression. It is shown that these sites are destined to form new root primordia from pericycle cells of both adventitious and main roots. Dose-dependent induction of root meristems by auxin overcomes the limitations of this naturally stochastic process and makes lateral root initiation amenable to biochemical analysis. Quiescent pericycle cells, which are developmentally arrested in the G₂ phase of the cell cycle, rapidly progress into M phase upon mitogenic stimulation. Colchicine and nocodazole, which block completion of mitosis, inhibited the activation of the HRGPnt3 promoter but did not block auxin induction of parA, a marker for de-differentiation in leaf mesophyll cell-derived protoplasts. Hydroxyurea, which inhibits cell-cycle progression at the G₁/S-phase transition and also blocks lateral root initiation, did not inhibit HRGPnt3 induction. Thus, HRGPnt3 induction precedes completion of the first cell division during primordium formation, and is one of the initial steps in a sequential program of gene expression activated upon stimulation of cell division for the development of a new meristem during lateral root initiation.     

Lateral root initiation: one step at a time

Plant growth relies heavily on a root system that is hidden belowground, which develops post-embryonically through the formation of lateral roots. The de novo formation of lateral root organs requires tightly coordinated asymmetric cell division of a limited number of pericycle cells located at the xylem pole. This typically involves the formation of founder cells, followed by a number of cellular changes until the cells divide and give rise to two unequally sized daughter cells. Over the past few years, our knowledge of the regulatory mechanisms behind lateral root initiation has increased dramatically. Here, I will summarize these recent advances, focusing on the prominent role of auxin and cell cycle activity, and elaborating on the three key steps of pericycle cell priming, founder cell establishment and asymmetric cell division. Taken together, recent findings suggest a tentative model in which successive auxin response modules are crucial for lateral root initiation, and additional factors provide more layers of control.     

THE ROOT APEX OF MALVA SYLVESTRIS. III. LATERAL ROOT DEVELOPMENT AND THE QUIESCENT CENTER

Both histological and autoradiographic procedures were used to follow lateral root initiation and development. Lateral roots of M. sylvestris were initiated in the pericycle, and although the endodermis became multiseriate, endodermal derivatives were not incorporated into the lateral root primordium. Apical organization of pre-emergent roots, characterized by two tiers of cortical initials, did not change with growth. During pre-emergent development there was no evidence of cortical lysogeny or quiescent center formation. Quiescent centers were present in both secondary and tertiary roots longer than 0.5 cm.     

Auxin transport promotes Arabidopsis lateral root initiation

Lateral root development in Arabidopsis provides a model for the study of hormonal signals that regulate postembryonic organogenesis in higher plants. Lateral roots originate from pairs of pericycle cells, in several cell files positioned opposite the xylem pole, that initiate a series of asymmetric, transverse divisions. The auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) arrests lateral root development by blocking the first transverse division(s). We investigated the basis of NPA action by using a cell-specific reporter to demonstrate that xylem pole pericycle cells retain their identity in the presence of the auxin transport inhibitor. However, NPA causes indoleacetic acid (IAA) to accumulate in the root apex while reducing levels in basal tissues critical for lateral root initiation. This pattern of IAA redistribution is consistent with NPA blocking basipetal IAA movement from the root tip. Characterization of lateral root development in the shoot meristemless1 mutant demonstrates that root basipetal and leaf acropetal auxin transport activities are required during the initiation and emergence phases, respectively, of lateral root development.     

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.