Isolation and characterization of cDNA clones corresponding with mRNAs that accumulate during auxin-induced lateral root formation

Lateral root formation in root cultures of Arabidopsis thaliana can be initiated by exogenous addition of auxin. In order to find cDNA clones of which the corresponding mRNAs accumulate during this process, a cDNA library was constructed from root cultures treated with the active auxin 1-naphthaleneacetic acid (1- NAA). Differential screening of this library with cDNA probes derived from mRNA populations isolated from root cultures treated with 1-NAA and the inactive analogue 2-naphthaleneacetic acid (2-NAA) led to the isolation of four cDNA clones, designated AIR1, AIR3, AIR9 and AIR12. Accumulation of the mRNAs starts between 4 and 8 h and continues till at least 24 h after addition of an active auxin. Sequence analysis revealed that AIR1 encodes a protein that is related to a large family of proteins that consist of a proline-rich or glycine- rich N-terminus and a hydrophobic, possibly membrane spanning C- terminus. The putative function of these proteins is coupling of the cell wall to the plasma membrane. Surprisingly, AIR1 lacks the proline-rich or glycine-rich N-terminus which is thought to be important for interaction with the cell wall. AIR3 encodes a subtilisin-like serine protease which is believed to be active outside the plant cell. Although AIR9 and AIR12 do not show any significant homology to sequences in the database, they are also predicted to function outside the cell. Our screening thus indicates that a variety of genes encoding extracellular proteins are activated during auxin-induced lateral root formation.     

LRP1, a gene expressed in lateral and adventitious root primordia of arabidopsis.

We describe a gene that is expressed in lateral and adventitious root primordia of Arabidopsis. The gene was identified by expression of a transposon-borne promoterless beta-glucuronidase gene in lateral root primordia. The gene, designated LRP1 for lateral root primordium 1, and its corresponding cDNA were cloned and sequenced. The expression pattern of the gene in lateral root primordia was confirmed by in situ hybridization with LRP1 cDNA probes. The LRP1 gene encodes a novel protein. LRP1 expression is activated during the early stages of root primordium development and is turned off prior to the emergence of lateral roots from the parent root. Insertion of the transposon in the LRP1 gene disrupted its expression. To evaluate the homozygous insertion line for a mutant phenotype, several aspects of wild-type lateral root development were analyzed. A mutant phenotype has not yet been identified in the insertion line; however, there is evidence that the gene belongs to a small gene family. LRP1 provides a molecular marker to study the early stages of lateral and adventitious root primordium development.     

Root system architecture in Arabidopsis grown in culture is regulated by sucrose uptake in the aerial tissues

This article presents a detailed model for the regulation of lateral root formation in Arabidopsis thaliana seedlings grown in culture. We demonstrate that direct contact between the aerial tissues and sucrose in the growth media is necessary and sufficient to promote emergence of lateral root primordia from the parent root. Mild osmotic stress is perceived by the root, which then sends an abscisic acid-dependent signal that causes a decrease in the permeability of aerial tissues; this reduces uptake of sucrose from the culture media, which leads to a repression of lateral root formation. Osmotic repression of lateral root formation in culture can be overcome by mutations that cause the cuticle of a plant's aerial tissues to become more permeable. Indeed, we report here that the previously described lateral root development2 mutant overcomes osmotic repression of lateral root formation because of a point mutation in Long Chain Acyl-CoA Synthetase2, a gene essential for cutin biosynthesis. Together, our findings (1) impact the interpretation of experiments that use Arabidopsis grown in culture to study root system architecture; (2) identify sucrose as an unexpected regulator of lateral root formation; (3) demonstrate mechanisms by which roots communicate information to aerial tissues and receive information in turn; and (4) provide insights into the regulatory pathways that allow plants to be developmentally plastic while preserving the essential balance between aboveground and belowground organs.     

Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings

Lateral root formation is profoundly affected by auxins. Here we present data which indicate that light influences the formation of indole-3-acetic acid (IAA) in germinating Arabidopsis seedlings. IAA transported from the developing leaves to the root system is detectable as a short-lived pulse in the roots and is required for the emergence of the lateral root primordia (LRP) during early seedling development. LRP emergence is inhibited by the removal of apical tissues prior to detection of the IAA pulse in the root, but this treatment has minimal effects on LRP initiation. Our results identify the first developing true leaves as the most likely source for the IAA required for the first emergence of the LRP, as removal of cotyledons has only a minor effect on LRP emergence in contrast to removal of the leaves. A basipetal IAA concentration gradient with high levels of IAA in the root tip appears to control LRP initiation, in contrast to their emergence. A significant increase in the ability of the root system to synthesize IAA is observed 10 days after germination, and this in turn is reflected in the reduced dependence of the lateral root emergence on aerial tissue-derived auxin at this stage. We propose a model for lateral root formation during early seedling development that can be divided into two phases: (i) an LRP initiation phase dependent on a root tip-localized IAA source, and (ii) an LRP emergence phase dependent on leaf-derived IAA up to 10 days after germination.     

Rhizobia can induce nodules in white clover by "hijacking" mature cortical cells activated during lateral root development

We examined a range of responses of root cortical cells to Rhizobium sp. inoculation to investigate why rhizobia preferentially nodulate legume roots in the zone of emerging root hairs, but generally fail to nodulate the mature root. We tested whether the inability to form nodules in the mature root is due to a lack of plant flavonoids to induce the bacterial genes required for nodulation or a failure of mature cortical cells to respond to Rhizobium spp. When rhizobia were inoculated in the zone of emerging root hairs, changes in beta-glucuronidase (GUS) expression from an auxin-responsive promoter (GH3), expression from three chalcone synthase promoters, and the accumulation of specific flavonoid compounds occurred in cortical cells prior to nodule formation. Rhizobia failed to induce these responses when inoculated in the mature root, even when co-inoculated with nod gene-inducing flavonoids. However, mature root hairs remained responsive to rhizobia and could support infection thread formation. This suggests that a deficiency in signal transduction is the reason for nodulation failure in the mature root. However, nodules could be initiated in the mature root at sites of lateral root emergence. A comparison between lateral root and nodule formation showed that similar patterns of GH3:gusA expression, chalcone synthase gene expression, and accumulation of a particular flavonoid compound occurred in the cortical cells involved in both processes. The results suggest that rhizobia can "hijack" cortical cells next to lateral root emergence sites because some of the early responses required for nodule formation have already been activated by the plant in those cells.     

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.     

Isolating the Arabidopsis thaliana genes for de novo purine synthesis by suppression of Escherichia coli mutants. I. 5'-Phosphoribosyl-5-aminoimidazole synthetase.

We have initiated an investigation of the de novo purine nucleotide biosynthetic pathway in the plant Arabidopsis thaliana. Functional suppression of Escherichia coli auxotrophs allowed the direct isolation of expressed Arabidopsis leaf cDNAs. Using this approach we have successfully suppressed mutants in 4 of the 12 genes in this pathway. One of these cDNA clones, encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase (PUR5) has been characterized in detail. Analysis of genomic DNA suggests that the Arabidopsis genome contains a single AIR synthetase gene. Analysis of the cDNA sequence and mRNA size suggests that this enzyme activity is encoded by a monofunctional polypeptide, similar to that of bacteria and unlike other eukaryotes. The Arabidopsis AIR synthetase contains a basic hydrophobic transit peptide consistent with transport into chloroplasts. Comparison of both the predicted amino acid and nucleotide sequence from Arabidopsis to those of eight other distant organisms suggests that the plant sequence is more similar to the bacterial sequences than to other eukaryotic sequences. This study provides the groundwork for future investigations into the regulation of de novo purine biosynthesis in plants. Additionally, we have demonstrated that functional suppression of bacterial mutants may provide a useful method for cloning a variety of plant genes.     

Mutations in a gene for plastid ribosomal protein S6-like protein reveal a novel developmental process required for the correct organization of lateral root meristem in Arabidopsis

Arabidopsis rfc3 mutants have previously been isolated with an altered fatty acid composition of membrane lipids. In this study, rfc3 was found to have a sucrose-conditional defect in the patterning of distal elements in the lateral root meristem. By utilizing this feature, a sucrose-sensitive process important for lateral root development was localized to the growing portion of rfc3 primary root. Because lateral root formation occurs at a later stage, this finding suggests the existence of an RFC3-dependent, non-primordium autonomous signal playing a role in the organization of lateral root meristem. Map-based cloning of RFC3 gene revealed that it encodes a plastid-localized ribosomal protein S6-like protein and provides a potential link between control of plastid gene expression and LR development.     

Arabidopsis AIR12 influences root development

Arabidopsis AUXIN INDUCED IN ROOTS (AIR 12) is a predicted to encode a glycosylphosphatidylinositol tail anchored protein. It has been associated with extracellular redox processes, but little is known about its physiological role. An air12 mutant line demonstrated increased germination rates in the presence of a range of abiotic stress factors and hormones, but not in the presence of ABA. Disruption of AIR12 also affected primary and lateral root development and was linked to changes in root catalase activity and superoxide production. We suggest AIR12 is an extracellular constituent linking both hormone and reactive oxygen signaling in plants.     

Regulation of Arabidopsis root development by nitrate availability

When the root systems of many plant species are exposed to a localized source of nitrate (NO3- they respond by proliferating their lateral roots to colonize the nutrient-rich zone. This study reviews recent work with Arabidopsis thaliana in which molecular genetic approaches are being used to try to understand the physiological and genetic basis for this response. These studies have led to the conclusion that there are two distinct pathways by which NO3- modulates root branching in Arabidopsis. On the one hand, meristematic activity in lateral root tips is stimulated by direct contact with an enriched source of NO3- (the localized stimulatory effect). On the other, a critical stage in the development of the lateral root (just after its emergence from the primary root) is highly susceptible to inhibition by a systemic signal that is related to the amount of NO3- absorbed by the plant (the systemic inhibitory effect). Evidence has been obtained that the localized stimulatory effect is a direct effect of the NO3- ion itself rather than a nutritional effect. A NO3(-)-inducible MADS-box gene (ANR1) has been identified which encodes a component of the signal transduction pathway linking the external NO3- supply to the increased rate of lateral root elongation. Experiments using auxin-resistant mutants have provided evidence for an overlap between the auxin and NO3- response pathways in the control of lateral root elongation. The systemic inhibitory effect, which does not affect lateral root initiation but delays the activation of the lateral root meristem, appears to be positively correlated with the N status of the plant and is postulated to involve a phloem-mediated signal from the shoot.     

Antisense Expression of an Arabidopsis Ran Binding Protein Renders Transgenic Roots Hypersensitive to Auxin and Alters Auxin-Induced Root Growth and Development by Arresting Mitotic Progress

We cloned a cDNA encoding an Arabidopsis Ran binding protein, AtRanBP1c, and generated transgenic Arabidopsis expressing the antisense strand of the AtRanBP1c gene to understand the in vivo functions of the Ran/RanBP signal pathway. The transgenic plants showed enhanced primary root growth but suppressed growth of lateral roots. Auxin significantly increased lateral root initiation and inhibited primary root growth in the transformants at 10 pM, several orders of magnitude lower than required to induce these responses in wild-type roots. This induction was followed by a blockage of mitosis in both newly emerged lateral roots and in the primary root, ultimately resulting in the selective death of cells in the tips of both lateral and primary roots. Given the established role of Ran binding proteins in the transport of proteins into the nucleus, these findings are consistent with a model in which AtRanBP1c plays a key role in the nuclear delivery of proteins that suppress auxin action and that regulate mitotic progress in root tips.     

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.     

Sites and regulation of auxin biosynthesis in Arabidopsis roots

Auxin has been shown to be important for many aspects of root development, including initiation and emergence of lateral roots, patterning of the root apical meristem, gravitropism, and root elongation. Auxin biosynthesis occurs in both aerial portions of the plant and in roots; thus, the auxin required for root development could come from either source, or both. To monitor putative internal sites of auxin synthesis in the root, a method for measuring indole-3-acetic acid (IAA) biosynthesis with tissue resolution was developed. We monitored IAA synthesis in 0.5- to 2-mm sections of Arabidopsis thaliana roots and were able to identify an important auxin source in the meristematic region of the primary root tip as well as in the tips of emerged lateral roots. Lower but significant synthesis capacity was observed in tissues upward from the tip, showing that the root contains multiple auxin sources. Root-localized IAA synthesis was diminished in a cyp79B2 cyp79B3 double knockout, suggesting an important role for Trp-dependent IAA synthesis pathways in the root. We present a model for how the primary root is supplied with auxin during early seedling development.     

Characterization of CYCLOPHILLIN38 shows that a photosynthesis-derived systemic signal controls lateral root emergence

Photosynthesis in leaves generates fixed-carbon resources and essential metabolites that support sink tissues, such as roots. Two of these metabolites, sucrose and auxin, promote growth in root systems, but the explicit connection between photosynthetic activity and control of root architecture has not been explored. Through a mutant screen to identify pathways regulating root system architecture, we identified a mutation in the Arabidopsis thaliana CYCLOPHILIN 38 (CYP38) gene, which causes accumulation of pre-emergent stage lateral roots. CYP38 was previously reported to stabilize photosystem II (PSII) in chloroplasts. CYP38 expression is enriched in shoots, and grafting experiments show that the gene acts non-cell-autonomously to promote lateral root emergence. Growth of wild-type plants under low-light conditions phenocopies the cyp38 lateral root emergence defect, as does the inhibition of PSII-dependent electron transport or Nicotinamide adenine dinucleotide phosphate (NADPH) production. Importantly, these perturbations to photosynthetic activity rapidly suppress lateral root emergence, which is separate from their effects on shoot size. Supplementary exogenous sucrose largely rescued primary root (PR) growth in cyp38, but not lateral root growth. Auxin (indole-3-acetic acid (IAA)) biosynthesis from tryptophan is dependent on reductant generated during photosynthesis. Consistently, we found that wild-type seedlings grown under low light and cyp38 mutants have highly diminished levels of IAA in root tissues. IAA treatment rescued the cyp38 lateral root defect, revealing that photosynthesis promotes lateral root emergence partly through IAA biosynthesis. These data directly confirm the importance of CYP38-dependent photosynthetic activity in supporting root growth, and define the specific contributions of two metabolites in refining root architecture under light-limited conditions.

Lateral root emergence is regulated via systemic signaling that incorporates photosynthesis-dependent redox control and auxin biosynthesis.     

Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses

We used the cytokinin-responsive Arabidopsis response regulator (ARR)5 gene promoter fused to a beta-glucuronidase (GUS) reporter gene, and cytokinin oxidase (CKX) genes from Arabidopsis thaliana (AtCKX3) and maize (ZmCKX1) to investigate the roles of cytokinins in lateral root formation and symbiosis in Lotus japonicus. ARR5 expression was undetectable in the dividing initial cells at early stages of lateral root formation, but later we observed high expression in the base of the lateral root primordium. The root tip continues to express ARR5 during subsequent development of the lateral root. These results suggest a dynamic role for cytokinin in lateral root development. We observed ARR5 expression in curled/deformed root hairs, and also in nodule primordia in response to Rhizobial inoculation. This expression declined once the nodule emerged from the parent root. Root penetration and migration of root-knot nematode (RKN) second-stage larvae (L2) did not elevate ARR5 expression, but a high level of expression was induced when L2 reached the differentiating vascular bundle and during early stages of the nematode-plant interaction. ARR5 expression was specifically absent in mature giant cells (GCs), although dividing cells around the GCs continued to express this reporter. The same pattern was observed using a green fluorescent protein (GFP) reporter driven by the ARR5 promoter in tomato. Overexpression of CKX genes rendered the transgenic hairy roots resistant to exogenous application of the cytokinin [N6-(Delta2 isopentenyl) adenine riboside] (iPR). CKX roots have significantly more lateral roots, but fewer nodules and nematode-induced root galls per plant, than control hairy roots.     

MM31/EIR1 promotes lateral root formation in Arabidopsis

Lateral root formation in Arabidopsis provides a model for the study of auxin function. Tryptophan (Trp) is a precursor of the auxin indoleacetic acid (IAA). To study the physiological function of Trp in auxin-related phenotypes, we examined the effect of Trp on lateral root formation. We found that Trp treatment enhanced lateral root formation and, by screening for mutants in which the effect of Trp on lateral root formation was enhanced, we isolated the mm31 mutant. Based on genetic and physiological analyses, we propose that MM31/EIR1 modulates lateral root formation by regulating the IAA polar transport system, and that auxin transport from the shoot to the root regulates lateral root formation.Key words: lateral root formation, Arabidopsis, EIR1, IAA, auxin     

The tobacco Cel7 gene promoter is auxin-responsive and locally induced in nematode feeding sites of heterologous plants

Emerging evidence suggests that plant cell-wall-modifying enzymes induced by root-parasitic nematodes play important roles in feeding cell formation. We previously identified a tobacco endo-β-1,4-glucanase (cellulase) gene, NtCel7 , that was strongly induced in both root-knot and cyst nematode feeding cells. To characterize further the developmental and nematode-responsive regulation of NtCel7 , we isolated the NtCel7 promoter and analysed its expression over a time course of nematode infection and in response to auxin, gibberellin, ethylene and sucrose in soybean and tomato hairy roots and in Arabidopsis containing the NtCel7 promoter fused to the β-glucuronidase (GUS) reporter gene. Histochemical analyses of transgenic plant materials revealed that the NtCel7 promoter exhibited a unique organ-specific expression pattern during plant development suggestive of important roles for NtCel7 in both vegetative and reproductive growth. In all plant species tested, strong GUS expression was observed in root tips and lateral root primordia of uninfected roots with weaker expression in the root vasculature. Further analyses of transgenic Arabidopsis plants revealed expression in shoot and root meristems and the vasculature of most organs during plant development. We also determined that the NtCel7 promoter was induced by auxin, but not gibberellin, ethylene or sucrose. Moreover, strong GUS activity was observed in both cyst and root-knot nematode-induced feeding sites in transgenic roots of soybean, tomato and Arabidopsis. The conserved developmental and nematode-responsive expression of the NtCel7 promoter in heterologous plants indicates that motifs of this regulatory element play a fundamental role in regulating NtCel7 gene expression within nematode feeding sites and that this regulation may be mediated by auxin.