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.     

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.     

Cytokinin inhibits lateral root initiation but stimulates lateral root elongation in rice (Oryza sativa)

Research in lateral root (LR) development mainly focuses on the role of auxin. This article reports the effect of cytokinins (kinetin and trans-zeatin) on LR formation in rice (Oryza sativa L.). Our results showed that cytokinin has an inhibitory effect on LR initiation and stimulatory effect on LR elongation. Both KIN and ZEA at a concentration of 1 microM and above completely inhibited lateral root primordium (LRP) formation. The inhibitory effect of cytokinin on LR initiation required a continuous presence of KIN or ZEA in the growth solution. Cytokinin did not show any inhibitory effect on LR emergence from the seminal root once LRPs had been formed. The LRPs that developed in cytokinin-free solution can emerge normally in the solution containing inhibitory concentration (1 microM) of KIN and ZEA. The KIN and ZEA treatment dramatically stimulated LR elongation at all the concentrations tested. Maximum LR elongation was observed at a concentration of 0.01 microM KIN and 0.001 microM ZEA. The epidermal cell length increased significantly in LRs of cytokinin treated seedlings compared to those of untreated control. This result indicates that the stimulation of LR elongation by cytokinin is due to increased cell length. Exogenously applied auxin counteracted the effect of cytokinin on LR initiation and LR elongation, suggesting that cytokinin acts on LR elongation through an auxin dependent pathway.     

An abscisic acid-sensitive checkpoint in lateral root development of Arabidopsis

Lateral root (LR) formation displays considerable plasticity in response to developmental and environmental signals. The mechanism whereby plants incorporate diverse regulatory signals into the developmental programme of LRs remains to be elucidated. Current concepts of lateral root regulation focus on the role of auxin. In this study, we show that another plant hormone, abscisic acid (ABA), also plays a critical role in the regulation of this post-embryonic developmental event. In the presence of exogenous ABA, LR development is inhibited. This occurs at a specific developmental stage, i.e. immediately after the emergence of the LR primordium (LRP) from the parent root and prior to the activation of the LR meristem, and is reversible. Interestingly, this inhibition requires 10-fold less ABA than the inhibition of seed germination and is only slightly reduced in characterised abi mutants, suggesting that it may involve novel ABA signalling mechanisms. We also present several lines of evidence to support the conclusion that the ABA-induced lateral root inhibition is mediated by an auxin-independent pathway. First, the inhibition could not be rescued by either exogenous auxin application or elevated auxin synthesis. Secondly, a mutation in the ALF3 gene, which is believed to encode an important component in the auxin-dependent regulatory pathway for the post-emergence LR development, does not affect the sensitivity of LRs to ABA. Thirdly, ABA and the alf3-1 mutation do not act at the same developmental point. To summarise, these results demonstrate a novel ABA-sensitive, auxin-independent checkpoint for lateral root development in Arabidopsis at the post-emergence stage. In addition, we also present data indicating that regulation of this developmental checkpoint may require novel ABA signalling mechanisms and that ABA suppresses auxin response in the LRPs.     

Developmental anatomy and auxin response of lateral root formation in Ceratopteris richardii

The homosporous fern Ceratopteris richardii exhibits a homorhizic root system where roots originate from the shoot system. These shoot-borne roots form lateral roots (LRs) that arise from the endodermis adjacent to the xylem poles, which is in contrast to flowering plants where LR formation arises from cell division in the pericycle. A detailed study of the fifth shoot-borne root showed that one lateral root mother cell (LRMC) develops in each two out of three successive merophytes. As a result, LRs emerge alternately in two ranks from opposite positions on a parent root. From LRMC initiation to LR emergence, three developmental stages were identified based on anatomical criteria. The addition of auxins (either indole-3-acetic acid or indole-3-butyric acid) to the growth media did not induce additional LR formation, but exogenous applications of both auxins inhibited parent root growth rate. Application of the polar auxin-transport inhibitor N-(1-naphthyl)phthalamic acid (NPA) also inhibited parent root growth without changing the LR initiation pattern. The results suggest that LR formation does not depend on root growth rate per se. The result that exogenous auxins do not promote LR formation in C. richardii is similar to reports for certain species of flowering plants, in which there is an acropetal LR population and the formation of the LRs is insensitive to the application of auxins. It also may indicate that different mechanisms control LR development in non-seed vascular plants compared with angiosperms, taking into consideration the long and independent evolutionary history of the two groups.     

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.     

Cytokinin-mediated cell cycling arrest of pericycle founder cells in lateral root initiation of Arabidopsis

In Arabidopsis, lateral root formation is a post-embryonic developmental event, which is regulated by hormones and environmental signals. In this study, via analyzing the expression of cyclin genes during lateral root (LR) formation, we report that cytokinins (CTKs) inhibit the initiation of LR through blocking the pericycle founder cells cycling at the G(2) to M transition phase, while the promotion by CTK of LR elongation is due to the stimulation of the G(1) to S transition. No significant difference was detected in the inhibitory effect of CTK on LR formation between wild-type plants and mutants defective in auxin response or transport. In addition, exogenously applied auxin at different concentrations could not rescue the CTK-mediated inhibition of LR initiation. Our data suggest that CTK and auxin might control LR initiation through two separate signaling pathways in Arabidopsis. The CTK-mediated repression of LR initiation is transmitted through the two-component signal system and mediated by the receptor CRE1.     

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.     

Regulation of lateral root formation in lettuce (Lactuca sativa) seedling roots: Interacting effects of α-naphthaleneacetic acid and kinetin

Growth substances, α-naphthaleneacetic (NAA) and kinetin, had an important role in the regulation of lateral root (LR) formation in lettuce ( Lactuca sativa L. cv. Grand Rapids) seedling roots. NAA (10^-5 M ) was a potent stimulator of LR initiation and caused a 600% increase in the number of lateral root primordia (LRP) compared to untreated roots. NAA was required for only the first 20 h of the 72 h treatment period for maximum stimulation of LRP initiation. Kinetin (2 × 10^-5 M ) effectively prevented the spontaneous formation of LRP and inhibited the NAA-stimulated production of LRP. Kinetin inhibition was maximal during the first 20 h of NAA treatment and this effect was not overcome by subsequent supply of NAA. Also, lettuce roots were most sensitive to kinetin at 20 h of NAA treatment, when the first signs of cell division were observed in the pericycle.     

An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin in pericycle cell activation

Arabidopsis (Arabidopsis thaliana) plants display a number of root developmental responses to low phosphate availability, including primary root growth inhibition, greater formation of lateral roots, and increased root hair elongation. To gain insight into the regulatory mechanisms by which phosphorus (P) availability alters postembryonic root development, we performed a mutant screen to identify genetic determinants involved in the response to P deprivation. Three low phosphate-resistant root lines (lpr1-1 to lpr1-3) were isolated because of their reduced lateral root formation in low P conditions. Genetic and molecular analyses revealed that all lpr1 mutants were allelic to BIG, which is required for normal auxin transport in Arabidopsis. Detailed characterization of lateral root primordia (LRP) development in wild-type and lpr1 mutants revealed that BIG is required for pericycle cell activation to form LRP in both high (1 mm) and low (1 microm) P conditions, but not for the low P-induced alterations in primary root growth, lateral root emergence, and root hair elongation. Exogenously supplied auxin restored normal lateral root formation in lpr1 mutants in the two P treatments. Treatment of wild-type Arabidopsis seedlings with brefeldin A, a fungal metabolite that blocks auxin transport, phenocopies the root developmental alterations observed in lpr1 mutants in both high and low P conditions, suggesting that BIG participates in vesicular targeting of auxin transporters. Taken together, our results show that auxin transport and BIG function have fundamental roles in pericycle cell activation to form LRP and promote root hair elongation. The mechanism that activates root system architectural alterations in response to P deprivation, however, seems to be independent of auxin transport and BIG.     

Auxin-dependent regulation of lateral root positioning in the basal meristem of Arabidopsis

In plants, the developmental mechanisms that regulate the positioning of lateral organs along the primary root are currently unknown. We present evidence on how lateral root initiation is controlled in a spatiotemporal manner in the model plant Arabidopsis thaliana. First, lateral roots are spaced along the main axis in a regular left-right alternating pattern that correlates with gravity-induced waving and depends on AUX1, an auxin influx carrier essential for gravitropic response. Second, we found evidence that the priming of pericycle cells for lateral root initiation might take place in the basal meristem, correlating with elevated auxin sensitivity in this part of the root. This local auxin responsiveness oscillates with peaks of expression at regular intervals of 15 hours. Each peak in the auxin-reporter maximum correlates with the formation of a consecutive lateral root. Third, auxin signaling in the basal meristem triggers pericycle cells for lateral root initiation prior to the action of INDOLE-3-ACETIC ACID14 (SOLITARY ROOT).     

The jasmonate receptor COI1 plays a role in jasmonate-induced lateral root formation and lateral root positioning in Arabidopsis thaliana

Jasmonic acid (JA) regulates a broad range of plant defense and developmental responses. COI1 has been recently found to act as JA receptor. In this report, we show that low micromolar concentrations of JA inhibited primary root (PR) growth and promoted lateral root (LR) formation in Arabidopsis wild-type (WT) seedlings. It was observed that the coi1-1 mutant was less sensitive to JA on pericycle cell activation to induce lateral root primordia (LRP) formation and presented alterations in lateral root positioning and lateral root emergence on bends. To investigate JA-auxin interactions important for remodeling of root system (RS) architecture, we tested the expression of auxin-inducible markers DR5:uidA and BA3:uidA in WT and coi1-1 seedlings in response to indole-3-acetic acid (IAA) and JA and analyzed the RS architecture of a suite of auxin-related mutants under JA treatments. We found that JA did not affect DR5:uidA and BA3:uidA expression in WT and coi1-1 seedlings. Our data also showed that PR growth inhibition in response to JA was likely independent of auxin signaling and that the induction of LRP required ARF7, ARF19, SLR, TIR1, AFB2, AFB3 and AXR1 loci. We conclude that JA regulation of postembryonic root development involves both auxin-dependent and independent mechanisms.     

Auxin signaling modulates LATERAL ROOT PRIMORDIUM1 (LRP1) expression during lateral root development in Arabidopsis

Auxin signaling mediated by various auxin/indole‐3‐acetic acid (Aux/IAAs) and AUXIN RESPONSE FACTORs (ARFs) regulate lateral root (LR) development by controlling the expression of downstream genes. LATERAL ROOT PRIMORDIUM1 (LRP1), a member of the SHORT INTERNODES/STYLISH (SHI/STY) family, was identified as an auxin‐inducible gene. The precise developmental role and molecular regulation of LRP1 in root development remain to be understood. Here we show that LRP1 is expressed in all stages of LR development, besides the primary root. The expression of LRP1 is regulated by histone deacetylation in an auxin‐dependent manner. Our genetic interaction studies showed that LRP1 acts downstream of auxin responsive Aux/IAAs‐ARFs modules during LR development. We showed that auxin‐mediated induction of LRP1 is lost in emerging LRs of slr‐1 and arf7arf19 mutants roots. NPA treatment studies showed that LRP1 acts after LR founder cell specification and asymmetric division during LR development. Overexpression of LRP1 (LRP1 OE) showed an increased number of LR primordia (LRP) at stages I, IV and V, resulting in reduced emerged LR density, which suggests that it is involved in LRP development. Interestingly, LRP1‐induced expression of YUC4, which is involved in auxin biosynthesis, contributes to the increased accumulation of endogenous auxin in LRP1 OE roots. LRP1 interacts with SHI, STY1, SRS3, SRS6 and SRS7 proteins of the SHI/STY family, indicating their possible redundant role during root development. Our results suggested that auxin and histone deacetylation affect LRP1 expression and it acts downstream of LR forming auxin response modules to negatively regulate LRP development by modulating auxin homeostasis in Arabidopsis thaliana.     

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.     

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.     

Involvement of G1-to-S transition and AhAUX-dependent auxin transport in abscisic acid-induced inhibition of lateral root primodia initiation in Arachis hypogaea L

Previous study indicated that increasing endogenous abscisic acid (ABA) level could inhibit the lateral root (LR) formation of peanuts. In this study, we investigated the mechanisms by which ABA regulated lateral root primordia (LRP) initiation in peanuts (Arachis hypogaea L). Results suggested that ABA inhibited LRP initiation through blocking G1-to-S transition in seedlings and mature roots: e.g. 5.8% increase in the proportion of G1 phase and 18% decrease in the proportion of S phase after ABA treatment for 6 days. Further study of the expression of the cell cycle marker gene for G2-to-M transition in peanut roots suggested that AhCYCB1 expression was regulated by ABA. We also investigated the cooperative regulation of LRP initiation by ABA and indole-3-acetic acid (IAA). ABA treatment greatly reduced the effects of endogenous IAA on mature roots. The expression of the IAA polar transport gene AhAUX1 appeared to be regulated by ABA since ABA inhibited auxin-mediated LRP initiation by suppressing AhAUX-dependent auxin transport in peanut roots. We further examined the effect of ABA on the expression of DR5::GUS and AtAUX1 in the model plant Arabidopsis. The results of Arabidopsis were consistent with that of the peanut.     

Effects of three auxin-inducible LBD members on lateral root formation in Arabidopsis thaliana

In Arabidopsis, two AUXIN RESPONSE FACTORs (ARF7 and ARF19) and several Aux/IAAs regulate auxin-induced lateral root (LR) formation. As direct targets of ARF7 and ARF19, LATERAL ORGAN BOUNDARIES DOMAIN 16 (LBD16), LBD29, and LBD18 have a biological function in the formation of lateral roots (LRs). However, the details of the functions of these three LBDs have remained unclear. Each single T-DNA insert mutant has been shown to have slightly fewer LRs than the wild type. We then created a triple mutant, which exhibited a dramatic defect in the LR formation. Our results show that the lbd mutations can lead to impairment in auxin-induced pericycle cell division and in the expression levels of some D-type cyclins (CYCDs). Simultaneously, PLETHORA (PLT) and PIN-FORMED (PIN), which have been well documented to promote cell mitotic activity and are required for auxin response effects, were down-regulated by these lbd mutations. Our results so far indicate that CYCDs, PLT, and PINs are the main targets of the LBDs. We believe that these three LBDs are involved in cell cycle progression of the pericycle in response to auxin. Overexpression of any of these three LBD genes in the triple mutant was found incapable of completely replacing the other two LBDs. The phenotypes of lbd29 mutants were not completely consistent with lbd16 or lbd18 mutants. This indicates that LBD29 may play a distinctive role compared with LBD16 or LBD18 and LBDs might play partially independent roles during the formation of LRs.