Development Of Root Primordia Research Articles

Do DEEPER ROOTING 1 Homologs Regulate the Lateral Root Slope Angle in Cucumber (Cucumis sativus)?

The architecture of the root system is fundamental to plant productivity. The rate of root growth, the density of lateral roots, and the spatial structure of lateral and adventitious roots determine the developmental plasticity of the root system in response to changes in environmental conditions. One of the genes involved in the regulation of the slope angle of lateral roots is DEEPER ROOTING 1 (DRO1). Its orthologs and paralogs have been identified in rice, Arabidopsis, and several other species. However, nothing is known about the formation of the slope angle of lateral roots in species with the initiation of lateral root primordia within the parental root meristem. To address this knowledge gap, we identified orthologs and paralogs of the DRO1 gene in cucumber (Cucumis sativus) using a phylogenetic analysis of IGT protein family members. Differences in the transcriptional response of CsDRO1, CsDRO1-LIKE1 (CsDRO1L1), and CsDRO1-LIKE2 (CsDRO1L2) to exogenous auxin were analyzed. The results showed that only CsDRO1L1 is auxin-responsive. An analysis of promoter-reporter fusions demonstrated that the CsDRO1, CsDRO1L1, and CsDRO1L2 genes were expressed in the meristem in cell files of the central cylinder, endodermis, and cortex; the three genes displayed different expression patterns in cucumber roots with only partial overlap. A knockout of individual CsDRO1, CsDRO1L1, and CsDRO1L2 genes was performed via CRISPR/Cas9 gene editing. Our study suggests that the knockout of individual genes does not affect the slope angle formation during lateral root primordia development in the cucumber parental root.

Interspecies co-expression analysis of lateral root development using inducible systems in rice, Medicago, and Arabidopsis.

Lateral roots are crucial for plant growth and development, making them an important target for research aiming to improve crop yields and food security. However, their endogenous ontogeny and, as it were, stochastic appearance challenge their study. Lateral Root Inducible Systems (LRIS) can be used to overcome these challenges by inducing lateral roots massively and synchronously. The combination of LRISs with transcriptomic approaches significantly advanced our insights in the molecular control of lateral root formation, in particular for Arabidopsis. Despite this success, LRISs have been underutilized for other plant species or for lateral root developmental stages later than the initiation. In this study, we developed and/or adapted LRISs in rice, Medicago, and Arabidopsis to perform RNA-sequencing during time courses that cover different developmental stages of lateral root formation and primordium development. As such, our study provides three extensive datasets of gene expression profiles during lateral root development in three different plant species. The three LRISs are highly effective but timing and spatial distribution of lateral root induction vary among the species. Detailed characterization of the stages in time and space in the respective species enabled an interspecies co-expression analysis to identify conserved players involved in lateral root development, as illustrated for the AUX/IAA and LBD gene families. Overall, our results provide a valuable resource to identify potentially conserved regulatory mechanisms in lateral root development, and as such will contribute to a better understanding of the complex regulatory network underlying lateral root development.

Anatomical observations on formation and development of adventitious root primordium in canes of Vitis sp.

Understanding the anatomical aspects of adventitious root primordium formation can provide valuable insights into the improvement of propagation techniques, rootstock selection, and overall vineyard management practices in Vitis spp. This work was carried out to investigate anatomical root primordium formation in the rooted cuttings of Cabernet Sauvignon (CS, Vitis vinifera L.) and the rootstock Kober 5BB (Vitis berlandieri x V. riparia) with their relationship to stem anatomy. One-node cuttings were grown under temperature-controlled conditions for 8 weeks. After removal of the roots and calli, the stem parts were fixed in a fixative solution. A revised method of safranin staining was applied to the 90 µm thick cross-sections made with a hand microtome. It was observed that root primordia were derived from the two different regions of the cane tissues: from the groups of cells close to the outside of the conductive tissue system and from the cell groups in the deeper site, close to the pith. Cultivars showed significant differences in terms of the regions where they had their root primordial initials. Number of potential root primordia was statistically higher in CS. Both CS and 5BB had root angles in the range of 83° to 86°. It was concluded that grapevine cuttings had only induced root primordia and the capacity to produce them was dependent on the genotype. Formation and development of root primordia and the anatomical differentiation of the cell groups were similar in Cabernet Sauvignon and 5BB.

Identification and function of miRNA-mRNA interaction pairs during lateral root development of hemi-parasitic Santalum album L. seedlings

Sandalwood (Santalum album L.) is a hemi-parasitic tree species famous for its santalol and santalene, which are extracted from its heartwood and roots. The ability to understand root functionality within its branched root system would benefit the regulation of sandalwood growth and enhance the commercial value of sandalwood. Phenotypic and anatomical evidence in this study indicated that seed germination stage 4 (SG4) seemed pivotal for lateral root (LR) morphogenesis. Small RNA (sRNA) high-throughput sequencing of root tissues at three sub-stages of SG4 (lateral root primordia initiation (LRPI), lateral root primordia development (LRPD), and lateral root primordia emergence (LRPE)) was performed to identify microRNAs (miRNAs) associated with LR development. A total of 135 miRNAs, including 70 differentially expressed miRNAs (DEMs), were screened. Ten DEMs were selected to investigate transcript abundance in different organs or developmental stages. Among 100 negative DEM-mRNA interaction pairs, four targets (Sa-miR166m_2, 408d, 858a, and novel_Sa-miR8) were selected for studying cleavage sites by 5′ RLM-RACE validation. The expression mode of the four miRNA-mRNA pairs was investigated after indole-3-acetic acid (IAA) treatment. IAA enhanced the abundance of homeobox-leucine-zipper protein 32 (HOX32), laccase 12 (LAC12), myeloblastosis86 (MYB86), and pectin methylesterase inhibitor6 (PMEI6) target transcripts by reducing the expression of Sa-miR166m_2, 408d, 858a, and novel_Sa-miR8 in the first 10 min. A schematic model of miRNA-regulated LR development is proposed for this hemi-parasitic species. This novel genetic information for improving sandalwood root growth and development may allow for the cultivation of fast-growing and high-yielding plantations.

Auxin Distribution in Lateral Root Primordium Development Affects the Size and Lateral Root Diameter of Rice.

Lateral roots (LRs) occupy a large part of the root system and play a central role in plant water and nutrient uptake. Monocot plants, such as rice, produce two types of LRs: the S-type (short and thin) and the L-type (long, thick, and capable of further branching). Because of the ability to produce higher-order branches, the L-type LR formation contributes to efficient root system expansion. Auxin plays a major role in regulating the root system development, but its involvement in developing different types of LRs is largely unknown. Here, we show that auxin distribution is involved in regulating LR diameter. Dynamin-related protein (DRP) genes were isolated as causative genes of the mutants with increased L-type LR number and diameter than wild-type (WT). In the drp mutants, reduced endocytic activity was detected in rice protoplast and LRs with a decreased OsPIN1b-GFP endocytosis in the protoplast. Analysis of auxin distribution using auxin-responsive promoter DR5 revealed the upregulated auxin signaling in L-type LR primordia (LRP) of the WT and the mutants. The application of polar auxin transport inhibitors enhanced the effect of exogenous auxin to increase LR diameter with upregulated auxin signaling in the basal part of LRP. Inducible repression of auxin signaling in the mOsIAA3-GR system suppressed the increase in LR diameter after root tip excision, suggesting a positive role of auxin signaling in LR diameter increase. A positive regulator of LR diameter, OsWOX10, was auxin-inducible and upregulated in the drp mutants more than the WT, and revealed as a potential target of ARF transcriptional activator. Therefore, auxin signaling upregulation in LRP, especially at the basal part, induces OsWOX10 expression, increasing LR diameter.

PUCHI represses early meristem formation in developing lateral roots of Arabidopsis thaliana.

Lateral root organogenesis is a key process in the development of a plant’s root system and its adaptation to the environment. During lateral root formation, an early phase of cell proliferation first produces a four-cell-layered primordium, and only from this stage onwards is a root meristem-like structure, expressing root stem cell niche marker genes, being established in the developing organ. Previous studies reported that the gene regulatory network controlling lateral root formation is organized into two subnetworks whose mutual inhibition may contribute to organ patterning. PUCHI encodes an AP2/ERF transcription factor expressed early during lateral root primordium development and required for correct lateral root formation. To dissect the molecular events occurring during this early phase, we generated time-series transcriptomic datasets profiling lateral root development in puchi-1 mutants and wild types. Transcriptomic and reporter analyses revealed that meristem-related genes were expressed ectopically at early stages of lateral root formation in puchi-1 mutants. We conclude that, consistent with the inhibition of genetic modules contributing to lateral root development, PUCHI represses ectopic establishment of meristematic cell identities at early stages of organ development. These findings shed light on gene network properties that orchestrate correct timing and patterning during lateral root formation.

Glutathione produced by γ-glutamyl cysteine synthetase acts downstream of hydrogen to positively influence lateral root branching

Hydrogen gas (H2) mediation of lateral root (LR) branching was previously described.However, related signaling pathway is largely unexplored. In this study, we discovered that application with H2 using hydrogen-rich water, mimicking the responses of exogenous glutathione (GSH), not only enhanced GSH synthesis, but also induced tomato LR development. The changes in the transcripts of auxin signaling-related genes and cell cycle regulatory genes were matched with above phenotypes. The addition of H2 could trigger higher transcript levels of SlGSH1 and SlGSH2, encoding γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS), confirming the stimulation of GSH synthesis. These responses were greatly abolished when the inhibitor of γ-ECS was applied. The inhibition in lateral root primordium development, especially in emergence stage, was also observed. Genetic evidence revealed that the defects in GSH production and lateral rooting in Arabidopsis cad2-1, a γ-ECS defective mutant, were obviously abolished in the presence of GSH compared to those in the presence of H2. Further evidence revealed that mRNA levels of target genes elicited by H2 in wild-type, were differentially impaired in mutant plants. Together, above data clearly demonstrated that γ-ECS-dependent GSH production might be closely associated with H2 control of LR branching.

Genetic variations of root development traits under different concentrations and forms of nitrogen in Bangladeshi rice (Oryza sativa L.) accessions

ABSTRACT Genetic variations in the root traits of rice remain unexplored in relation to varietal groups and diverse ecosystems. Therefore, we evaluated 257 Bangladeshi accessions, which included landraces and improved varieties belonging to four ecotypes (Aus, Aman, Boro and Jhum), to understand genetic variations in the total root length (TRL), maximum root length and root number under different concentrations of ammonium and nitrate nitrogen in hydroponic culture. There were large variations in all traits across all nitrogen levels. Active elongation of individual roots of the landrace type across all nitrogen conditions suggested a higher potential for the uptake of nutrients by extending roots distribution in any nitrogen environment, while the active development of primary root primordia in the improved type in the presence of nitrate suggested a higher potential for the uptake of nitrate from the soil surface by extending roots distribution around soil surface. Cluster analysis classified the examined accessions into two groups according to their TRL: cluster I with a longer TRL and cluster II with a shorter TRL across all nitrogen conditions. Each of these groups was further divided into two sub-clusters: sub-cluster Ia, in which the longer TRL resulted from active development of primary root primordia, sub-cluster Ib, in which the longer TRL resulted from active elongation of individual roots, sub-cluster IIa, in which the shorter TRL resulted from severe reduction of the elongation of individual roots, and sub-cluster IIb, in which the shorter TRL resulted from a moderate reduction in the elongation of individual roots. Further investigation revealed a difference in the composition of these four sub-clusters among the four ecotypes, indicating that Aman was the most diverse ecotype, followed by Aus. The root traits of these promising accessions could be exploited in the future to understand genetic and physiological mechanism(s) of adaptation to diverse ecosystems.

Gene expression profiling reveals the effects of light on adventitious root formation in lotus seedlings (Nelumbo nucifera Gaertn.)

BackgroundLotus is an aquatic horticultural crop that is widely cultivated in most regions of China and is used as an important off-season vegetable. The principal root of lotus is degenerated, and adventitious roots (ARs) are irreplaceable for plant growth. We found that no ARs formed under darkness and that exposure to high-intensity light significantly promoted the development of root primordia. Four differential expression libraries based on three light intensities were constructed to monitor metabolic changes, especially in indole-3-acetic acid (IAA) and sugar metabolism.ResultsAR formation was significantly affected by light, and high light intensity accelerated AR development. Metabolic changes during AR formation under different light intensities were evaluated using gene expression profiling by high-throughput tag-sequencing. More than 2.2 × 104 genes were obtained in each library; the expression level of most genes was between 0.01 and 100 (FPKF value). Libraries constructed from plants grown under darkness (D/CK), under 5000 lx (E/CK), and under 20,000 lx (F/CK) contained 1739, 1683, and 1462 upregulated genes and 1533, 995, and 834 downregulated genes, respectively, when compared to those in the initial state (CK). Additionally, we found that 1454 and 478 genes had altered expression in a comparison of libraries D/CK and F/CK. Gene transcription between libraries D/F ranged from a 5-fold decrease to a 5-fold increase. Twenty differentially expressed genes (DEGs) were involved in the signal transduction pathway, 28 DEGs were related to the IAA response, and 35 DEGs were involved in sugar metabolism. We observed that the IAA content was enhanced after seed germination, even in darkness; this was responsible for AR formation. We also observed that sucrose could eliminate the negative effect of 150 μMol IAA during AR development.ConclusionsAR formation was regulated by IAA, even in the dark, where induction and developmental processes could also be completed. In addition, 36 genes displayed altered expression in carbohydrate metabolism and ucrose metabolism was involved in AR development (expressed stage) according to gene expression and content change characteristics.

Endogenous Hypoxia in Lateral Root Primordia Controls Root Architecture by Antagonizing Auxin Signaling in Arabidopsis

As non-photosynthesizing organs, roots are dependent on diffusion of oxygen from the external environment and, in some instances, from the shoot for their aerobic metabolism. Establishment of hypoxic niches in the developing tissues of plants has been postulated as a consequence of insufficient diffusion of oxygen to satisfy the demands throughout development. Here, we report that such niches are established at specific stages of lateral root primordia development in Arabidopsis thaliana grown under aerobic conditions. Using gain- and loss-of-function mutants, we show that ERF-VII transcription factors, which mediate hypoxic responses, control root architecture by acting in cells with a high level of auxin signaling. ERF-VIIs repress the expression of the auxin-induced genes LBD16, LBD18, and PUCHI, which are essential for lateral root development, by binding to their promoters. Our results support a model in which the establishment of hypoxic niches in the developing lateral root primordia contributes to the shutting down of key auxin-induced genes and regulates the production of lateral roots.

The Influence of Nanoparticles of Biogenic Ferrihydrite on the Rooting of Lignified Cuttings of the Ledebour Willow

The influence of nanoparticles of biogenic ferrihydrite on the root formation of lignified cuttings of the Ledebour willow (Salix ledebouriana Trautv.) was investigated. The rooting of stem cuttings was performed in water using a phytohormone (indoyl-3-acetic acid) and nanoparticles. In comparison with the variants of incubation of cuttings in water and in a solution containing indolyl-3-acetic acid, the number and total length of the adventitious roots after 5 and 15 days of rooting were greatest in the cuttings treated with nanoparticles. In the early period of root development, increased levels of free radicals and intensities of the hyperfine structure lines of Mn2+ cleavage were recorded in the EPR spectra of plant tissues (lenticels of cuttings of treated with nanoparticles). The latter is related to the activity of manganese-containing superoxide dismutase. The response of cuttings to the treatment with nanoparticles was recorded by EPR before manifestation of adventitious root primordium initiation and development.

Hydrogen sulfide acts downstream of methane to induce cucumber adventitious root development

Previous results have shown that hydrogen sulfide (H2S), mainly catalyzed by l-cysteine desulfhydrase (DES) in plants, triggers adventitious rooting. The objective of this study was to test whether H2S is involved in methane (CH4)-induced adventitious root development in cucumber explants. First, we observed that the activities of DES, endogenous H2S production, and thereafter adventitious root development were induced by CH4 and NaHS (an H2S donor). Some responses were sensitive to hypotaurine (HT; a scavenger of H2S), showing that endogenous H2S production and adventitious rooting were obviously blocked. The development of adventitious root primordia was also impaired. Further molecular evidence revealed that CH4-induced gene expression of CsDNAJ-1, CsCDPK1, CsCDPK5, CsCDC6 (a cell-division-related gene), CsAux22D-like, and CsAux22B-like (two auxin-signaling genes), several molecular markers responsible for adventitious rooting, were blocked by the co-treatment with HT. The occurrence of CH4-elicited S-sulfhydration during the above responses was also sensitive to the removal of endogenous H2S, suggesting the requirement of H2S. Taken together, our results reveal a vital role of endogenous H2S in CH4-triggered cucumber adventitious root development, and thus provide a comprehensive window into the complex signaling transduction pathway in CH4-mediated root organogenesis.

An auxin-induced β-type endo-1,4-β-glucanase in poplar is involved in cell expansion and lateral root formation.

PtrGH9A7, a poplar β-type endo-1,4-β-glucanase gene induced by auxin, promotes both plant growth and lateral root development by enhancing cell expansion. Endo-1,4-β-glucanase (EGase) family genes function in multiple aspects of plant growth and development. Our previous study found that PtrCel9A6, a poplar EGase gene of the β subfamily, is specifically expressed in xylem tissue and is involved in the cellulose biosynthesis required for secondary cell wall formation (Yu et al. in Mol Plant 6:1904-1917, 2013). To further explore the functions and regulatory mechanism of β-subfamily EGases, we cloned and characterized another poplar β-type EGase gene PtrGH9A7, a close homolog of PtrCel9A6. In contrast to PtrCel9A6, PtrGH9A7 is predominantly expressed in parenchyma tissues of the above-ground part; in roots, PtrGH9A7 expression is specifically restricted to lateral root primordia at all stages from initiation to emergence and is strongly induced by auxin application. Heterologous overexpression of PtrGH9A7 promotes plant growth by enhancing cell expansion, suggesting a conserved role for β-type EGases in 1,4-β-glucan chains remodeling, which is required for cell wall loosening. Moreover, the overexpression of PtrGH9A7 significantly increases lateral root number, which might result from improved lateral root primordium development due to enhanced cell expansion. Taken together, these results demonstrate that this β-type EGase induced by auxin signaling has a novel role in promoting lateral root formation as well as in enhancing plant growth.

Histological characterization of the lateral root primordium development in rice.

BackgroundLateral root constitutes an important part of root system either in tap root plants or fibrous root plants. The development of lateral root primordium (LRP) in Arabidopsis, which has a tap root system with simple radial structure of primary root, has been well characterized. However, limited knowledge has been acquired on the plants with fibrous root system, such as rice. This is mainly due to their multiple cell layers coated on root, which disturb the observation of LRP.ResultsWe used an easy and quick method to strip the epidermal and cortex tissues of primary root so that the LRP can be easily observed under microscope. In this way, we observed the developmental processes of LRP in rice. In addition, we described the expression dynamics of several root development related genes, especially OsPINs (PIN-FORWMED), during the process of LRP development.ConclusionsWe reported an easy and quick method for LRP observation in rice and suggested a “fountain” model of auxin transport in LRP of rice, which is similar with that in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-014-0042-x) contains supplementary material, which is available to authorized users.

High efficient transformation of auxin reporter gene into trifoliate orange via Agrobacterium rhizogenes-mediated co-transformation

An efficient Agrobacterium rhizogenes mediated transformation system of Poncirus trifoliata was developed. The combination of A. rhizogenes MSU440 with epicotyl segments was confirmed to be a suitable system for genetic transformation in P. trifoliata. About 3 weeks after inoculation with bacteria, hairy roots emerged at the cut surfaces of epicotyl segments on Murashige and Tucker selective medium free of plant growth regulators. The presence of rol B, β-glucuronidase (GUS) and yellow fluorescent protein (YFP) genes in co-transformed hairy roots was confirmed by fluorescence observation, GUS staining and polymerase chain reaction (PCR) analysis. The co-transformation rate based on GUS staining was as high as 68.3 %. A regeneration procedure of transgenic plantlets from hairy roots was also established. Out of 45 hairy root segments from different co-transformed lines, 23 were successfully regenerated into rooted plantlets. The presence and expression of YFP gene in the regenerated co-transformed plants were further confirmed by Southern blot hybridization and quantitative reverse transcription PCR. The DR5∷GUS–YFP reporter system was used to study the auxin response maxima at the apices of primary hairy roots and their lateral roots. Auxin distribution and response maxima during lateral root primordium development were also studied. The hairy roots obtained by A. rhizogenes mediated transformation were confirmed to be a good system for investigating root development in P. trifoliata. Furthermore, the genetic transformation and plant regeneration system via A. rhizogenes provides an alternative method for genetic engineering in Citrus.

Characterization of a Small Auxin-Up RNA (SAUR)-Like Gene Involved in Arabidopsis thaliana Development

The root of Arabidopsis thaliana is used as a model system to unravel the molecular nature of cell elongation and its arrest. From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a Small auxin-up RNA (SAUR)-like gene was found to be up regulated. As it appeared as the 76th gene in the family, it was named SAUR76. Root and leaf growth of overexpression lines ectopically expressing SAUR76 indicated the possible involvement of the gene in the division process. Using promoter::GUS and GFP lines strong expression was seen in endodermal and pericycle cells at the end of the elongation zone and during several stages of lateral root primordia development. ACC and IAA/NAA were able to induce a strong up regulation of the gene and changed the expression towards cortical and even epidermal cells at the beginning of the elongation zone. Confirmation of this up regulation of expression was delivered using qPCR, which also indicated that the expression quickly returned to normal levels when the inducing IAA-stimulus was removed, a behaviour also seen in other SAUR genes. Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane. SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.

To branch or not to branch: the role of pre-patterning in lateral root formation

The establishment of a pre-pattern or competence to form new organs is a key feature of the postembryonic plasticity of plant development, and the elaboration of such pre-patterns leads to remarkable heterogeneity in plant form. In root systems, many of the differences in architecture can be directly attributed to the outgrowth of lateral roots. In recent years, efforts have focused on understanding how the pattern of lateral roots is established. Here, we review recent findings that point to a periodic mechanism for establishing this pattern, as well as roles for plant hormones, particularly auxin, in the earliest steps leading up to lateral root primordium development. In addition, we compare the development of lateral root primordia with in vitro plant regeneration and discuss possible common molecular mechanisms.

Role of the Arabidopsis PIN6 auxin transporter in auxin homeostasis and auxin-mediated development.

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.

The AP2/EREBP Gene PUCHI Co-Acts with LBD16/ASL18 and LBD18/ASL20 Downstream of ARF7 and ARF19 to Regulate Lateral Root Development in Arabidopsis

The developmental process of lateral root formation consists of priming, initiation, primordium development and the emergence of lateral roots from the primary root. Molecular genetic studies with Arabidopsis have revealed several key transcriptional regulators involved in lateral root development. However, their functional interaction has not been fully characterized yet. Here we utilized a genetic approach to understand some of these interactions, revealing that PUCHI functioning in morphogenesis of early lateral root primordium is regulated downstream of ARF7/ARF19 and acts with LBD16(ASL18)/LBD18(ASL20) to regulate lateral root development. We showed that auxin-responsive expression of PUCHI was significantly reduced in arf7 or arf19 single mutants and completely abolished in arf7 arf19 double mutants. Consistent with this, β-glucuronidase (GUS) expression under the PUCHI promoter in arf7 arf19 was greatly reduced in the lateral root primordium compared with that in the wild type and did not respond to exogenous auxin. Results of GUS expression analyses under the PUCHI, LBD16 or LBD18 promoter in lbd16, lbd18 single and double mutants or puchi demonstrated that PUCHI and LBD16 or LBD18 do not regulate each other's expression. Lateral root phenotypes of double and triple mutants of lbd16, lbd18 and puchi showed that the puchi mutation in lbd16 and lbd18 mutants synergistically decreased the number of emerged lateral roots. These analyses also showed that puchi affected lateral root primordium development of lbd16 or lbd18 additively but differentially. Taken together, these results suggest that PUCHI co-acts with LBD16 and LBD18 to control lateral root primordium development and lateral root emergence.

Tissue organization of fasciated lateral roots of Arabidopsis mutants suggestive of the robust nature of outer layer patterning

In three temperature-sensitive mutants of Arabidopsis, root redifferentiation 1 (rrd1), rrd2, and root initiation defective 4 (rid4), formation of fasciated lateral roots was previously observed under high temperature conditions of 28°C. When lateral roots were induced from explants of very young seedlings of these mutants by culture with exogenously supplied auxin at 28°C, expansion of lateral root primordia leading to lateral root fasciation occurred reproducibly and semi-synchronously with a high frequency. This experimental system allowed us to examine how radial organization of root tissues is altered in association with expansion of primordia. Analysis with various tissue-specific reporter genes indicated that in the fasciated lateral roots, cell files of the stele are increased markedly while the numbers of cortical and epidermal cell layers are not changed. This suggests that radial organization during root primordium development involves a mechanism that makes outer layer patterning more robust than inner layer patterning against unusual enlargement of the morphogenetic field.