Shoot-supplied ammonium targets the root auxin influx carrier AUX1 and inhibits lateral root emergence in Arabidopsis

Deposition of ammonium (NH₄⁺) from the atmosphere is a substantial environmental problem. While toxicity resulting from root exposure to NH₄⁺ is well studied, little is known about how shoot‐supplied ammonium (SSA) affects root growth. In this study, we show that SSA significantly affects lateral root (LR) development. We show that SSA inhibits lateral root primordium (LRP) emergence, but not LRP initiation, resulting in significantly impaired LR number. We show that the inhibition is independent of abscisic acid (ABA) signalling and sucrose uptake in shoots but relates to the auxin response in roots. Expression analyses of an auxin‐responsive reporter, DR5:GUS, and direct assays of auxin transport demonstrated that SSA inhibits root acropetal (rootward) auxin transport while not affecting basipetal (shootward) transport or auxin sensitivity of root cells. Mutant analyses indicated that the auxin influx carrier AUX1, but not the auxin efflux carriers PIN‐FORMED (PIN)1 or PIN2, is required for this inhibition of LRP emergence and the observed auxin response. We found that AUX1 expression was modulated by SSA in vascular tissues rather than LR cap cells in roots. Taken together, our results suggest that SSA inhibits LRP emergence in Arabidopsis by interfering with AUX1‐dependent auxin transport from shoot to root.     

The auxin transporter,OsAUX1, is involved in primary root and root hair elongation and in Cd stress responses in rice (Oryza sativa L.)

Auxin and cadmium (Cd) stress play critical roles during root development. There are only a few reports on the mechanisms by which Cd stress influences auxin homeostasis and affects primary root (PR) and lateral root (LR) development, and almost nothing is known about how auxin and Cd interfere with root hair (RH) development. Here, we characterize rice osaux1 mutants that have a longer PR and shorter RHs in hydroponic culture, and that are more sensitive to Cd stress compared to wild‐type (Dongjin). OsAUX1 expression in root hair cells is different from that of its paralogous gene, AtAUX1, which is expressed in non‐hair cells. However, OsAUX1, like AtAUX1, localizes at the plasma membrane and appears to function as an auxin tranporter. Decreased auxin distribution and contents in the osaux1 mutant result in reduction of OsCyCB1;1 expression and shortened PRs, LRs and RHs under Cd stress, but may be rescued by treatment with the membrane‐permeable auxin 1‐naphthalene acetic acid. Treatment with the auxin transport inhibitors 1‐naphthoxyacetic acid and N‐1‐naphthylphthalamic acid increased the Cd sensitivity of WT rice. Cd contents in the osaux1 mutant were not altered, but reactive oxygen species‐mediated damage was enhanced, further increasing the sensitivity of the osaux1 mutant to Cd stress. Taken together, our results indicate that OsAUX1 plays an important role in root development and in responses to Cd stress.     

Osmotic regulation of root system architecture

Although root system architecture is known to be highly plastic and strongly affected by environmental conditions, we have little understanding of the underlying mechanisms controlling root system development. Here we demonstrate that the formation of a lateral root from a lateral root primordium is repressed as water availability is reduced. This osmotic-responsive regulatory mechanism requires abscisic acid (ABA) and a newly identified gene, LRD2. Mutant analysis also revealed interactions of ABA and LRD2 with auxin signaling. Surprisingly, further examination revealed that both ABA and LRD2 control root system architecture even in the absence of osmotic stress. This suggests that the same molecules that mediate responses to environmental cues can also be regulators of intrinsic developmental programs in the root system.     

Auxin reflux between the endodermis and pericycle promotes lateral root initiation

Lateral root (LR) formation is initiated when pericycle cells accumulate auxin, thereby acquiring founder cell (FC) status and triggering asymmetric cell divisions, giving rise to a new primordium. How this auxin maximum in pericycle cells builds up and remains focused is not understood. We report that the endodermis plays an active role in the regulation of auxin accumulation and is instructive for FCs to progress during the LR initiation (LRI) phase. We describe the functional importance of a PIN3 (PIN‐formed) auxin efflux carrier‐dependent hormone reflux pathway between overlaying endodermal and pericycle FCs. Disrupting this reflux pathway causes dramatic defects in the progress of FCs towards the next initiation phase. Our data identify an unexpected regulatory function for the endodermis in LRI as part of the fine‐tuning mechanism that appears to act as a check point in LR organogenesis after FCs are specified.     

Nitric oxide mediates strigolactone signaling in auxin and ethylene-sensitive lateral root formation in sunflower seedlings

Strigolactones (SLs) play significant role in shaping root architecture whereby auxin-SL crosstalk has been observed in SL-mediated responses of primary root elongation, lateral root formation and adventitious root (AR) initiation. Whereas GR24 (a synthetic strigolactone) inhibits LR and AR formation, the effect of SL biosynthesis inhibitor (fluridone) is just the opposite (root proliferation). Naphthylphthalamic acid (NPA) leads to LR proliferation but completely inhibits AR development. The diffusive distribution of PIN1 in the provascular cells in the differentiating zone of the roots in response to GR24, fluridone or NPA treatments further indicates the involvement of localized auxin accumulation in LR development responses. Inhibition of LR formation by GR24 treatment coincides with inhibition of ACC synthase activity. Profuse LR development by fluridone and NPA treatments correlates with enhanced [Ca²⁺]_cyt in the apical region and differentiating zones of LR, indicating a critical role of [Ca²⁺] in LR development in response to the coordinated action of auxins, ethylene and SLs. Significant enhancement of carotenoid cleavage dioxygenase (CCD) activity (enzyme responsible for SL biosynthesis) in tissue homogenates in presence of cPTIO (NO scavenger) indicates the role of endogenous NO as a negative modulator of CCD activity. Differences in the spatial distribution of NO in the primary and lateral roots further highlight the involvement of NO in SL-modulated root morphogenesis in sunflower seedlings. Present work provides new report on the negative modulation of SL biosynthesis through modulation of CCD activity by endogenous nitric oxide during SL-modulated LR development.     

Lateral root initiation in Arabidopsis: developmental window, spatial patterning, density and predictability

BACKGROUND AND AIMS: The basic regulatory mechanisms that control lateral root (LR) initiation are still poorly understood. An attempt is made to characterize the pattern and timing of LR initiation, to define a developmental window in which LR initiation takes place and to address the question of whether LR initiation is predictable. METHODS: The spatial patterning of LRs and LR primordia (LRPs) on cleared root preparations were characterized. New measures of LR and LRP densities (number of LRs and/or LRPs divided by the length of the root portions where they are present) were introduced and illustrate the shortcomings of the more customarily used measure through a comparative analysis of the mutant aux1-7. The enhancer trap line J0121 was used to monitor LR initiation in time-lapse experiments and a plasmolysis-based method was developed to determine the number of pericycle cells between successive LRPs. KEY RESULTS: LRP initiation occurred strictly acropetally and no de novo initiation events were found between already developed LRs or LRPs. However, LRPs did not become LRs in a similar pattern. The longitudinal spacing of lateral organs was variable and the distance between lateral organs was proportional to the number of cells and the time between initiations of successive LRPs. There was a strong tendency towards alternation in LR initiation between the two pericycle cell files adjacent to the protoxylem poles. LR density increased with time due to the emergence of slowly developing LRPs and appears to be unique for individual Arabidopsis accessions. CONCLUSIONS: In Arabidopsis there is a narrow developmental window for LR initiation, and no specific cell-count or distance-measuring mechanisms have been found that determine the site of successive initiation events. Nevertheless, the branching density and lateral organ density (density of LRs and LRPs) are accession-specific, and based on the latter density the average distance between successive LRs can be predicted.     

Cd胁迫下黄菖蒲幼苗根系生长与Cd积累的研究

采用水培法研究了Cd胁迫对黄菖蒲（Iris pseudacorus L．）幼苗根系生长和Cd含量的影响,用透射电镜观察了Cd胁迫对根尖细胞亚显微结构的影响并确定了Cd沉积部位。结果表明,10mg·L^-1 Cd处理对黄菖蒲幼苗根系的生长有一定促进作用,根毛数量及不定根上侧根的数量、密度和长度均高于对照;Cd浓度高于25mg·L^-1时,根系的生长发育受到抑制,上述指标均显著低于对照。用10～40mg·L^-1 Cd处理后,黄菖蒲幼苗不定根上侧根中的Cd含量均显著高于未长侧根的不定根和去掉侧根的不定根（P〈0．05）,说明不定根上的侧根对Cd沉积有重要作用。100mg·L^-1 Cd处理能导致根尖细胞质浓缩、质壁分离和液泡变大,且大量Cd颗粒沉积在细胞壁上和细胞间隙中,表明细胞壁和细胞间隙对Cd的沉积作用可能是黄菖蒲幼苗耐Cd胁迫的重要机理之一。     

Auxin-induced inhibition of lateral root initiation contributes to root system shaping in Arabidopsis thaliana

The hormone auxin is known to inhibit root elongation and to promote initiation of lateral roots. Here we report complex effects of auxin on lateral root initiation in roots showing reduced cell elongation after auxin treatment. In Arabidopsis thaliana, the promotion of lateral root initiation by indole-3-acetic acid (IAA) was reduced as the IAA concentration was increased in the nanomolar range, and IAA became inhibitory at 25 nM. Detection of this unexpected inhibitory effect required evaluation of root portions that had newly formed during treatment, separately from root portions that existed prior to treatment. Lateral root initiation was also reduced in the iaaM-OX Arabidopsis line, which has an endogenously increased IAA level. The ethylene signaling mutants ein2-5 and etr1-3, the auxin transport mutants aux1-7 and eir1/pin2, and the auxin perception/response mutant tir1-1 were resistant to the inhibitory effect of IAA on lateral root initiation, consistent with a requirement for intact ethylene signaling, auxin transport and auxin perception/response for this effect. The pericycle cell length was less dramatically reduced than cortical cell length, suggesting that a reduction in the pericycle cell number relative to the cortex could occur with the increase of the IAA level. Expression of the DR5:GUS auxin reporter was also less effectively induced, and the AXR3 auxin repressor protein was less effectively eliminated in such root portions, suggesting that decreased auxin responsiveness may accompany the inhibition. Our study highlights a connection between auxin-regulated inhibition of parent root elongation and a decrease in lateral root initiation. This may be required to regulate the spacing of lateral roots and optimize root architecture to environmental demands.     

Abscisic acid promotes pre-emergence stages of lateral root development in Medicago truncatula

The plant root system is important for plant anchorage and nutrition. Among the different characteristics of the root system, root branching is a major factor of plasticity and adaptation to changing environments. Indeed, many biotic and abiotic stresses, such as drought or symbiotic interactions, influence root branching. Many studies concerning root development and root branching were performed on the model plant Arabidopsis thaliana, but this model plant has a very simplified root structure and is not able to establish any symbiotic interactions. We have recently described 7 stages for lateral root development in the model legume Medicago truncatula and found significant differences in the tissular contribution of root cell layers to the formation of new lateral roots (LR). We have also described 2 transgenic lines expressing the DR5:GUS and DR5:VENUS-N7 reporter genes that are useful to follow LR formation at early developmental stages. Here, we describe the use of these transgenic lines to monitor LR developmental responses of M. truncatula to the phytohormone abscisic acid (ABA) which is a major actor of stress and symbiotic interactions. We show that ABA promotes the formation of new lateral root primordia and their development, mostly at the late, pre-emergence stage.     

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.     

OsORC3 is required for lateral root development in rice

The origin recognition complex (ORC) is a pivotal element in DNA replication, heterochromatin assembly, checkpoint regulation and chromosome assembly. Although the functions of the ORC have been determined in yeast and model animals, they remain largely unknown in the plant kingdom. In this study, Oryza sativa Origin Recognition Complex subunit 3 (OsORC3) was cloned using map‐based cloning procedures, and functionally characterized using a rice (Oryza sativa) orc3 mutant. The mutant showed a temperature‐dependent defect in lateral root (LR) development. Map‐based cloning showed that a G→A mutation in the 9th exon of OsORC3 was responsible for the mutant phenotype. OsORC3 was strongly expressed in regions of active cell proliferation, including the primary root tip, stem base, lateral root primordium, emerged lateral root primordium, lateral root tip, young shoot, anther and ovary. OsORC3 knockdown plants lacked lateral roots and had a dwarf phenotype. The root meristematic zone of ORC3 knockdown plants exhibited increased cell death and reduced vital activity compared to the wild‐type. CYCB1;1::GUS activity and methylene blue staining showed that lateral root primordia initiated normally in the orc3 mutant, but stopped growing before formation of the stele and ground tissue. Our results indicate that OsORC3 plays a crucial role in the emergence of lateral root primordia.     

RLF,a cytochrome b5‐like heme/steroid binding domain protein,controls lateral root formation independently of ARF7/19‐mediated auxin signaling in Arabidopsis thaliana

Lateral root (LR) formation is important for the establishment of root architecture in higher plants. Recent studies have revealed that LR formation is regulated by an auxin signaling pathway that depends on auxin response factors ARF7 and ARF19, and auxin/indole‐3‐acetic acid (Aux/IAA) proteins including SOLITARY‐ROOT (SLR)/IAA14. To understand the molecular mechanisms of LR formation, we isolated a recessive mutant rlf (reduced lateral root formation) in Arabidopsis thaliana. The rlf‐1 mutant showed reduction of not only emerged LRs but also LR primordia. Analyses using cell‐cycle markers indicated that the rlf‐1 mutation inhibits the first pericycle cell divisions involved in LR initiation. The rlf‐1 mutation did not affect auxin‐induced root growth inhibition but did affect LR formation over a wide range of auxin concentrations. However, the rlf‐1 mutation had almost no effect on auxin‐inducible expression of LATERAL ORGAN BOUNDARIES‐DOMAIN16/ASYMMETRIC LEAVES2‐LIKE18 (LBD16/ASL18) and LBD29/ASL16 genes, which are downstream targets of ARF7/19 for LR formation. These results indicate that ARF7/19‐mediated auxin signaling is not blocked by the rlf‐1 mutation. We found that the RLF gene encodes At5g09680, a protein with a cytochrome b₅‐like heme/steroid binding domain. RLF is ubiquitously expressed in almost all organs, and the protein localizes in the cytosol. These results, together with analysis of the genetic interaction between the rlf‐1 and arf7/19 mutations, indicate that RLF is a cytosolic protein that positively controls the early cell divisions involved in LR initiation, independent of ARF7/19‐mediated auxin signaling.     

Exogenous auxin-induced NO synthesis is nitrate reductase-associated in Arabidopsis thaliana root primordia

Nitric oxide (NO) functions in various physiological and developmental processes in plants. However, the source of this signaling molecule in the diversity of plant responses is not well understood. It is known that NO mediates auxin-induced adventitious and lateral root (LR) formation. In this paper, we provide genetic and pharmacological evidence that the production of NO is associated with the nitrate reductase (NR) enzyme during indole-3-butyric acid (IBA)-induced lateral root development in Arabidopsis thaliana L. NO production was detected using 4,5-diaminofluorescein diacetate (DAF-2DA) in the NR-deficient nia1, nia2 and Atnoa1 (former Atnos1) mutants of A. thaliana. An inhibitor for nitric oxide synthase (NOS) N(G)-monomethyl-l-arginine (l-NMMA) was applied. Our data clearly show that IBA increased LR frequency in the wild-type plant and the LR initials emitted intensive NO-dependent fluorescence of the triazol product of NO and DAF-2DA. Increased levels of NO were restricted only to the LR initials in contrast to primary root (PR) sections, where NO remained at the control level. The mutants had different NO levels in their control state (i.e. without IBA treatment): nia1, nia2 showed lower NO fluorescence than Atnoa1 or the wild-type plant. The role of NR in IBA-induced NO formation in the wild type was shown by the zero effects of the NOS inhibitors l-NMMA. Finally, it was clearly demonstrated that IBA was able to induce NO generation in both the wild-type and Atnoa1 plants, but failed to induce NO in the NR-deficient mutant. It is concluded that the IBA-induced NO production is nitrate reductase-associated during lateral root development in A. thaliana.     

The soybean root‐specific protein kinase GmWNK1 regulates stress‐responsive ABA signaling on the root system architecture

In humans, members of the WNK protein kinase family are osmosensitive regulators of cell volume homeostasis and epithelial ion transport, and mutation of these proteins causes a rare inherited form of hypertension due to increased renal NaCl re‐absorption. A related class of kinases was recently discovered in plants, but their functions are largely unknown. We have identified a root‐specific WNK kinase homolog, GmWNK1, in soybean (Glycine max). GmWNK1 expression was detected in the root, specifically in root cells associated with lateral root formation, and was down‐regulated by abscisic acid (ABA), as well as by mannitol, sucrose, polyethylene glycol and NaCl. In vitro and in vivo experiments showed that GmWNK1 interacts with another soybean protein, GmCYP707A1, which is a key ABA 8′‐hydroxylase that functions in ABA catabolism. Furthermore, 35S‐GmWNK1 transgenic soybean plants had reduced lateral root number and length compared with wild‐type, suggesting a role of GmWNK1 in the regulation of root system architecture. We propose that GmWNK1 functions to fine‐tune ABA‐dependent ABA homeostasis, thereby mediating the regulation of the root system architecture by ABA and osmotic signals. The study has revealed a new function of a plant WNK1 gene from the important staple crop soybean, and has identified a new component of a regulatory pathway that is involved not only in ABA signaling, but also in the repression of lateral root formation by an ABA‐dependent mechanism distinct from known ABA signaling pathways.     

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.     

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.     

Adventitious lateral rooting: the plasticity of root system architecture

Root formation under natural conditions is plastic in response to multiple signals. Recent studies suggested that the WUSCHEL‐RELATED HOMEOBOX11 (WOX11)‐mediated adventitious root formation pathway can occur in the primary root (PR) in Arabidopsis thaliana, resulting in the production of a specific type of lateral roots (LRs) in response to wounding or environmental signals. This process differs from the previously characterized process for LR development, which does not require WOX11. The WOX11‐mediated PR‐derived roots can be considered like LRs that exhibit an ‘adventitious’ feature. Therefore, we consider these roots to be adventitious lateral roots (adLRs). The identification of WOX11‐mediated adLRs implies that studying the formation of roots in response to wounding and environmental signals is important for characterizing the plasticity of the root system architecture.     

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.