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.     

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.     

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.     

GAL4-GFP enhancer trap lines for genetic manipulation of lateral root development in Arabidopsis thaliana

Lateral root development occurs throughout the life of the plant and is responsible for the plasticity of the root system. In Arabidopsis thaliana, lateral root founder cells originate from pericycle cells adjacent to xylem poles. In order to study the mechanisms of lateral root development, a population of Arabidopsis GAL4-GFP enhancer trap lines were screened and two lines were isolated with GAL4 expression in root xylem-pole pericycle cells (J0121), i.e. in cells competent to become lateral root founder cells, and in young lateral root primordia (J0192). These two enhancer trap lines are very useful tools with which to study the molecular and cellular bases of lateral root development using targeted gene expression. These lines were used for genetic ablation experiments by targeting the expression of a toxin-encoding gene. Moreover, the molecular bases of the enhancer trap expression pattern were characterized. These results suggest that the lateral-root-specific GAL4 expression pattern in J0192 is due to a strong enhancer in the promoter of the LOB-domain protein gene LBD16.     

Early primordium morphogenesis during lateral root initiation in Arabidopsis thaliana

The first morphogenetic events of lateral root primordium (LRP) formation in the Arabidopsis thaliana (L.) Heynh. pericycle occur soon after cells of the primary root complete elongation. Pericycle cells in direct contact with underlying protoxylem cells participate in LRP formation. Two types of LRP initiation were found, longitudinal uni- and bi-cellular. These occur when a single or two pericycle cells within a file, respectively, become founder cells for the entire longitudinal extent of the LRP. Histochemical and cytological analysis suggests that three is the minimum number of cells required to initiate an LRP. In young primordia comprising less than 32 cells, the average cell-doubling time was 3.7 h, indicating a drastic acceleration of cell cycle progression after lateral root initiation. Early in LRP development, cell growth is limited and therefore cytokinesis leads to a reduction of cell volume, similar to cleavage division cycles during animal and plant embryogenesis. The striking coordination of proliferation between pericycle cells in adjacent files in direct contact with the underlying protoxylem implies that intercellular signaling mechanisms act in the root apical meristem or later in development.     

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.     

β-glucuronidase as a marker for clonal analysis of tomato lateral roots

In higher plants, the root-shoot axis established during embryogenesis is extended and modified by the development of primary and lateral apical meristems. While the structure of several shoot apical meristems has been deduced by combining histological studies with clonal analysis, the application of this approach to root apical meristems has been limited by a lack of visible genetic markers. We have tested the feasibility of using a synthetic gene consisting of the maize transposable elementActivator (Ac) inserted between a 35S CaMV promoter and the coding region of a -glucuronidase (GUS) reporter gene as a means of marking cell lineages in roots. The GUS gene was activated in individual cells byAc excision, and the resulting sectors of GUS-expressing cells were detected with the histochemical stain X-Gluc. Sectors in lateral roots originated from bothAc excision in meristematic cells and from parent root sectors that bisect the founder cell population for the lateral root initial. Analysis of root tip sectors confirmed that the root cap, and root proper have separate initials. Large sectors in the body of the lateral root encompassed both cortex and vascular tissues. The number of primary initial cells predicted from the size and arrangement of the sectors observed ranged from two to four and appeared to vary between roots. We conclude that transposon-based clonal analysis using GUS expression as a genetic marker is an effective approach for deducing the functional organization of root apical meristems.     

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.     

Auxin minimum defines a developmental window for lateral root initiation

Root system architecture depends on lateral root (LR) initiation that takes place in a relatively narrow developmental window (DW). Here, we analyzed the role of auxin gradients established along the parent root in defining this DW for LR initiation. Correlations between auxin distribution and response, and spatiotemporal control of LR initiation were analyzed in Arabidopsis thaliana and tomato (Solanum lycopersicum). In both Arabidopsis and tomato roots, a well defined zone, where auxin content and response are minimal, demarcates the position of a DW for founder cell specification and LR initiation. We show that in the zone of auxin minimum pericycle cells have highest probability to become founder cells and that auxin perception via the TIR1/AFB pathway, and polar auxin transport, are essential for the establishment of this zone. Altogether, this study reveals that the same morphogen-like molecule, auxin, can act simultaneously as a morphogenetic trigger of LR founder cell identity and as a gradient-dependent signal defining positioning of the founder cell specification. This auxin minimum zone might represent an important control mechanism ensuring the LR initiation steadiness and the acropetal LR initiation pattern.     

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.     

Tissue‐specific inactivation by cytosine deaminase/uracil phosphoribosyl transferase as a tool to study plant biology

Recent advances in the study of plant developmental and physiological responses have benefited from tissue‐specific approaches, revealing the role of some cell types in these processes. Such approaches have relied on the inactivation of target cells using either toxic compounds or deleterious genes; however, both tissue‐specific and truly inducible tools are lacking in order to precisely target a developmental window or specific growth response. We engineered the yeast fluorocytosine deaminase (FCY1) gene by creating a fusion with the bacterial uracil phosphoribosyl transferase (UPP) gene. The recombinant protein converts the precursor 5‐fluorocytosine (5‐FC) into 5‐fluorouracyl, a drug used in the treatment of a range of cancers, which triggers DNA and RNA damage. We expressed the FCY‐UPP gene construct in specific cell types using enhancer trap lines and promoters, demonstrating that this marker acts in a cell‐autonomous manner. We also showed that it can inactivate slow developmental processes like lateral root formation by targeting pericycle cells. It also revealed a role for the lateral root cap and the epidermis in controlling root growth, a faster response. The 5‐FC precursor acts systemically, as demonstrated by its ability to inhibit stomatal movements when supplied to the roots in combination with a guard cell‐specific promoter. Finally, we demonstrate that the tissular inactivation is reversible, and can therefore be used to synchronize plant responses or to determine cell type‐specific functions during different developmental stages. This tool will greatly enhance our capacity to understand the respective role of each cell type in plant physiology and development.     

Repression of early lateral root initiation events by transient water deficit in barley and maize

The formation of lateral roots (LRs) is a key driver of root system architecture and developmental plasticity. The first stage of LR formation, which leads to the acquisition of founder cell identity in the pericycle, is the primary determinant of root branching patterns. The fact that initiation events occur asynchronously in a very small number of cells inside the parent root has been a major difficulty in the study of the molecular regulation of branching patterns. Inducible systems that trigger synchronous lateral formation at predictable sites have proven extremely valuable in Arabidopsis to decipher the first steps of LR formation. Here, we present a LR repression system for cereals that relies on a transient water-deficit treatment, which blocks LR initiation before the first formative divisions. Using a time-lapse approach, we analysed the dynamics of this repression along growing roots and were able to show that it targets a very narrow developmental window of the initiation process. Interestingly, the repression can be exploited to obtain negative control root samples where LR initiation is absent. This system could be instrumental in the analysis of the molecular basis of drought-responsive as well as intrinsic pathways of LR formation in cereals.     

The cell-cycle promoter cdc2aAt from Arabidopsis thaliana is induced in the lateral roots of the actinorhizal tree Allocasuarina verticillata during the early stages of the symbiotic interaction with Frankia

The symbiosis between the actinorhizal tree Allocasuarina verticillata and the actinomycete Frankia leads to the formation of root nodules inside which bacteria fix atmospheric nitrogen. Actinorhizal nodule organogenesis starts with the induction of cell divisions in the root cortex and in the pericycle cells opposite protoxylem poles near Frankia -infected root hairs. To study the ability of Frankia to induce progression through the cell cycle, we monitored the expression of the β-glucuronidase ( gus ) gene driven by the promoter from cdc2aAt , an Arabidopsis cyclin-dependent kinase gene that displays competence for cell division, during plant growth and nodule ontogenesis. In non-symbiotic tissues, the gus gene was mainly expressed in primary and secondary meristems of roots and shoots. Auxins and cytokinins were found to induce reporter gene activity in the root system of whole plants, showing that the promoter cdc2aAt displayed the same regulation by hormones in Allocasuarina as that reported in Arabidopsis . In transgenic nodules, gus expression was found to be restricted to the phellogen. During the early stages of the interaction between Frankia and the plant root system, cdc2aAt was strongly induced in the lateral roots surrounded by hyphae of the actinomycete. Histochemical analysis of β-glucuronidase activity revealed that cells from the pericycle opposite protoxylem poles were very deeply stained. These data indicate that upon Frankia infection, cells from the lateral roots, and notably pericycle cells that can give rise to a nodule or a root primordium, prepare to re-enter the cell cycle.     

A phylogenetic strategy based on a legume-specific whole genome duplication yields symbiotic cytokinin type-A response regulators

Legumes host their Rhizobium spp. symbiont in novel root organs called nodules. Nodules originate from differentiated root cortical cells that dedifferentiate and subsequently form nodule primordia, a process controlled by cytokinin. A whole-genome duplication has occurred at the root of the legume Papilionoideae subfamily. We hypothesize that gene pairs originating from this duplication event and are conserved in distinct Papilionoideae lineages have evolved symbiotic functions. A phylogenetic strategy was applied to search for such gene pairs to identify novel regulators of nodulation, using the cytokinin phosphorelay pathway as a test case. In this way, two paralogous type-A cytokinin response regulators were identified that are involved in root nodule symbiosis. Response Regulator9 (MtRR9) and MtRR11 in medicago (Medicago truncatula) and an ortholog in lotus (Lotus japonicus) are rapidly induced upon Rhizobium spp. Nod factor signaling. Constitutive expression of MtRR9 results in arrested primordia that have emerged from cortical, endodermal, and pericycle cells. In legumes, lateral root primordia are not exclusively formed from pericycle cells but also require the involvement of the root cortical cell layer. Therefore, the MtRR9-induced foci of cell divisions show a strong resemblance to lateral root primordia, suggesting an ancestral function of MtRR9 in this process. Together, these findings provide a proof of principle for the applied phylogenetic strategy to identify genes with a symbiotic function in legumes.