Lateral root development in the maize (Zea mays) lateral rootless1 mutant

Background and Aims

The maize lrt1 (lateral rootless1) mutant is impaired in its development of lateral roots during early post-embryonic development. The aim of this study was to characterize, in detail, the influences that the mutation exerts on lateral root initiation and the subsequent developments, as well as to describe the behaviour of the entire plant under variable environmental conditions.

Methods

Mutant lrt1 plants were cultivated under different conditions of hydroponics, and in between sheets of moist paper. Cleared whole mounts and anatomical sections were used in combination with both selected staining procedures and histochemical tests to follow root development. Root surface permeability tests and the biochemical quantification of lignin were performed to complement the structural data.

Key Results

The data presented suggest a redefinition of lrt1 function in lateral roots as a promoter of later development; however, neither the complete absence of lateral roots nor the frequency of their initiation is linked to lrt1 function. The developmental effects of lrt1 are under strong environmental influences. Mutant primordia are affected in structure, growth and emergence; and the majority of primordia terminate their growth during this last step, or shortly thereafter. The lateral roots are impaired in the maintenance of the root apical meristem. The primary root shows disturbances in the organization of both epidermal and subepidermal layers. The lrt1-related cell-wall modifications include: lignification in peripheral layers, the deposition of polyphenolic substances and a higher activity of peroxidase.

Conclusions

The present study provides novel insights into the function of the lrt1 gene in root system development. The lrt1 gene participates in the spatial distribution of initiation, but not in its frequency. Later, the development of lateral roots is strongly affected. The effect of the lrt1 mutation is not as obvious in the primary root, with no influences observed on the root apical meristem structure and maintenance; however, development of the epidermis and cortex are impaired.     

The growth defect of lrt1, a maize mutant lacking lateral roots, can be complemented by symbiotic fungi or high phosphate nutrition

The growth of three maize (Zea mays L.) mutants, each impaired in the formation of one individual element of its root system, was compared under "natural" limiting phosphate conditions (0.1 mM). Mutant plants exhibiting a reduction in root hairs (rth3-1) or a depletion of crown and brace roots (rtcs) grew as well as the corresponding wild-type plants. However, mutant plants lacking lateral roots (lrt1) showed a strong reduction in plant growth. The growth defect of lrt1 was overcome when it was grown in association with an arbuscular mycorrhizal fungus, Glomus mosseae. Establishment of symbiosis was associated with the occurrence of a new type of lateral root. These new lateral roots were stunted and highly branched, giving rise to a bush-like structure. Supply of high phosphate (1 microM) ameliorated the growth of lrt1 plants too, but less efficiently than the symbiosis did. Hence, arbuscular mycorrhizal fungi as well as phosphate functionally complemented the lrt1 mutation.     

Studies on sodium bypass flow in lateral rootless mutants lrt1 and lrt2, and crown rootless mutant crl1 of rice (Oryza sativa L.)

An apoplastic pathway, the so‐called bypass flow, is important for Na⁺ uptake in rice (Oryza sativa L.) under saline conditions; however, the precise site of entry is not yet known. We report the results of our test of the hypothesis that bypass flow of Na⁺ in rice occurs at the site where lateral roots emerge from the main roots. We investigated Na⁺ uptake and bypass flow in lateral rootless mutants (lrt1, lrt2), a crown rootless mutant (crl1), their wild types (Oochikara, Nipponbare and Taichung 65, respectively) and in seedlings of rice cv. IR36. The results showed that shoot Na⁺ concentration in lrt1, lrt2 and crl1 was lower (by 20–23%) than that of their wild types. In contrast, the bypass flow quantified using trisodium‐8‐hydroxy‐1,3,6‐pyrenetrisulphonic acid (PTS) was significantly increased in the mutants, from an average of 1.1% in the wild types to 3.2% in the mutants. Similarly, bypass flow in shoots of IR36 where the number of lateral and crown roots had been reduced through physical and hormonal manipulations was dramatically increased (from 5.6 to 12.5%) as compared to the controls. The results suggest that the path of bypass flow in rice is not at the sites of lateral root emergence.     

Isolation and characterization of rtcs, a maize mutant deficient in the formation of nodal roots

The root system of maize consists of the primary root and a variable number of lateral seminal-, crown- and brace roots. Except for the primary root and some minor roots forming at the mesocotyl, all other roots grow out of nodal regions, namely, the embryogenic scutellar node and the underground—as well as the lower above-ground stem nodes. Besides their role in water and nutrient uptake, some of these roots (crown- and brace roots) are essential for the lodging resistance of the plants. This property of the crown roots has now been successfully used for screening a segregating F₂ population of a cross between a flint inbred line and an En transposon line. Two allelic root-deficient mutants have been isolated and have been designated rtcs-1 and rtcs-2 for their complete lack of formation of c rown- and lateral s eminal roots. They survive by the ability of the primary root to support the growth of the developing plant. The monogenic and recessive mutants appear to be affected in an early root-forming function since no primordia are formed either in the case of embryo-borne lateral seminal or stem-derived crown roots. The Rtcs locus could be mapped to the short arm of chromosome 1 with the help of a co-segregating RAPD marker. The effect of the mutation seems to be highly specific since no pleiotropic effects on other parts of the plants have been observed. The formation of adventitious roots can, however, still be induced in the mesocotyl region of the mutant.     

Lateral roots affect the proteome of the primary root of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Lateral roots are initiated from the pericycle cells of other types of roots and remain in contact with these roots throughout their life span. Although this physical contact has the potential to permit the exchange of signals, little is known about the flow of information from the lateral roots to the primary root. To begin to study these interactions the proteome of the primary root system of the maize (Zea mays L.) lrt1 mutant, which does not initiate lateral roots, was compared with the corresponding proteome of wild-type seedlings 9 days after germination. Approximately 150 soluble root proteins were resolved by two-dimensional electrophoresis and analyzed by MALDI-ToF mass spectrometry and database searching. The 96 most abundant proteins from a pH 4–7 gradient were analyzed; 67 proteins representing 47 different Genbank accessions were identified. Interestingly, 10 (15/150) of the detected proteins were preferentially expressed in lrt1 roots that lack lateral roots. Eight of these lrt1-specific proteins were identified and four are involved in lignin metabolism. This study demonstrates for the first time the influence of lateral roots on the proteome of the primary root system. To our knowledge this is the first study to demonstrate an interaction between two plant organs (viz., lateral and primary roots) at the level of the proteome.     

Cooperative action of SLR1 and SLR2 is required for lateral root-specific cell elongation in maize

Lateral roots play an important role in water and nutrient uptake largely by increasing the root surface area. In an effort to characterize lateral root development in maize (Zea mays), we have isolated from Mutator (Mu) transposon stocks and characterized two nonallelic monogenic recessive mutants: slr1 and slr2 (short lateral roots1 and 2), which display short lateral roots as a result of impaired root cell elongation. The defects in both mutants act specifically during early postembryonic root development, affecting only the lateral roots emerging from the embryonic primary and seminal roots but not from the postembryonic nodal roots. These mutations have no major influence on the aboveground performance of the affected plants. The double mutant slr1; slr2 displays a strikingly different phenotype than the single mutants. The defect in slr1; slr2 does not only influence lateral root specific cell elongation, but also leads to disarranged cellular patterns in the primary and seminal roots. However, the phase-specific nature of the single mutants is retained in the double mutant, indicating that the two loci cooperate in the wild type to maintain the lateral root specificity during a short time of early root development.     

Genetic analysis of adventitious root formation with a novel series of temperature-sensitive mutants of Arabidopsis thaliana

When cultured on media containing the plant growth regulator auxin, hypocotyl explants of Arabidopsis thaliana generate adventitious roots. As a first step to investigate the genetic basis of adventitious organogenesis in plants, we isolated nine temperature-sensitive mutants defective in various stages in the formation of adventitious roots: five root initiation defective (rid1 to rid5) mutants failed to initiate the formation of root primordia; in one root primordium defective (rpd1) mutant, the development of root primordia was arrested; three root growth defective (rgd1, rgd2, and rgd3) mutants were defective in root growth after the establishment of the root apical meristem. The temperature sensitivity of callus formation and lateral root formation revealed further distinctions between the isolated mutants. The rid1 mutant was specifically defective in the reinitiation of cell proliferation from hypocotyl explants, while the rid2 mutant was also defective in the reinitiation of cell proliferation from root explants. These two mutants also exhibited abnormalities in the formation of the root apical meristem when lateral roots were induced at the restrictive temperature. The rgd1 and rgd2 mutants were deficient in root and callus growth, whereas the rgd3 mutation specifically affected root growth. The rid5 mutant required higher auxin concentrations for rooting at the restrictive temperature, implying a deficiency in auxin signaling. The rid5 phenotype was found to result from a mutation in the MOR1/GEM1 gene encoding a microtubule-associated protein. These findings about the rid5 mutant suggest a possible function of the microtubule system in auxin response.     

Environmental Regulation of Lateral Root Initiation in Arabidopsis

Plant morphology is dramatically influenced by environmental signals. The growth and development of the root system is an excellent example of this developmental plasticity. Both the number and placement of lateral roots are highly responsive to nutritional cues. This indicates that there must be a signal transduction pathway that interprets complex environmental conditions and makes the "decision" to form a lateral root at a particular time and place. Lateral roots originate from differentiated cells in adult tissues. These cells must reenter the cell cycle, proliferate, and redifferentiate to produce all of the cell types that make up a new organ. Almost nothing is known about how lateral root initiation is regulated or coordinated with growth conditions. Here, we report a novel growth assay that allows this regulatory mechanism to be dissected in Arabidopsis. When Arabidopsis seedlings are grown on nutrient media with a high sucrose to nitrogen ratio, lateral root initiation is dramatically repressed. Auxin localization appears to be a key factor in this nutrient-mediated repression of lateral root initiation. We have isolated a mutant, lateral root initiation 1 (lin1), that overcomes the repressive conditions. This mutant produces a highly branched root system on media with high sucrose to nitrogen ratios. The lin1 phenotype is specific to these growth conditions, suggesting that the lin1 gene is involved in coordinating lateral root initiation with nutritional cues. Therefore, these studies provide novel insights into the mechanisms that regulate the earliest steps in lateral root initiation and that coordinate plant development with the environment.     

The novel symbiotic phenotype of enhanced-nodulating mutant of Lotus japonicus: astray mutant is an early nodulating mutant with wider nodulation zone

We have isolated a novel enhanced-nodulating mutant astray (Ljsym77) from Lotus japonicus. The name astray derives from the non-symbiotic phenotype of this mutant, agravitropic lateral roots that go "astray" against gravity. In this report we evaluated the symbiotic aspects of this mutant in detail. The astray mutant developed approximately twice the number of nodules on a wider area of roots compared with the wild type. Furthermore, the astray mutant demonstrated early initiation of nodule development, which is an unprecedented symbiotic phenotype. The astray seedlings showed normal sensitivity to the general inhibitors of nodulation such as ethylene and nitrate. These results indicate that the astray mutant is distinct from the hypernodulating mutants reported previously, and that the ASTRAY gene acts as an early and negative regulator in the cascade of nodule development.     

MDR-like ABC transporter AtPGP4 is involved in auxin-mediated lateral root and root hair development

Previous data have suggested an involvement of MDR/PGP-like ABC transporters in transport of the plant hormone auxin and, recently, AtPGP1 has been demonstrated to catalyze the primary active export of auxin. Here we show that related isoform AtPGP4 is expressed predominantly during early root development. AtPGP4 loss-of-function plants reveal enhanced lateral root initiation and root hair lengths both known to be under the control of auxin. Further, atpgp4 plants show altered sensitivities toward auxin and the auxin transport inhibitor, NPA. Finally, mutant roots reveal elevated free auxin levels and reduced auxin transport capacities. These results together with yeast growth assays suggest a direct involvement of AtPGP4 in auxin transport processes controlling lateral root and root hair development.     

The effect of moisture stress on stolon and adventitious root development in white clover (Trifolium repens L.)

Summary Humidity, at the young nodes of white clover stolones, varied by enclosing nodes in the atmosphere above a range of saturated solutions, inhibited root initiation at 85% RH or less. The threshold humidity for root initiation increased to about 93% on young nodes subject to moisture stress or old nodes on well watered plants in which root initiation had been previously suppressed by low humidity.Roots at old nodes and at the three youngest on stolons were either subject to moisture stress or adequately watered. Growth of young roots and N₂-fixation were more adversely affected by the direct effects of drought than by subjecting old roots to drought. Although old roots under stress affected new root growth and N₂-fixation, length of roots and lateral root number were little affected. By contrast stolon growth was affected more by stress to old roots than to young nodes, although after 6 weeks the contribution made by young roots to stolon growth was almost as high as old roots.The data suggest that deep roots at old nodes will allow clover stolons to grow during drought due to the high acropetal movement of water but initiation of roots and functioning of young roots at the soil surface will be adversely affected, with possible implications on the persistence of clover.     

Crown rootless1, which is essential for crown root formation in rice, is a target of an AUXIN RESPONSE FACTOR in auxin signaling

Although the importance of auxin in root development is well known, the molecular mechanisms involved are still unknown. We characterized a rice (Oryza sativa) mutant defective in crown root formation, crown rootless1 (crl1). The crl1 mutant showed additional auxin-related abnormal phenotypic traits in the roots, such as decreased lateral root number, auxin insensitivity in lateral root formation, and impaired root gravitropism, whereas no abnormal phenotypic traits were observed in aboveground organs. Expression of Crl1, which encodes a member of the plant-specific ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES protein family, was localized in tissues where crown and lateral roots are initiated and overlapped with beta-glucuronidase staining controlled by the DR5 promoter. Exogenous auxin treatment induced Crl1 expression without de novo protein biosynthesis, and this induction required the degradation of AUXIN/INDOLE-3-ACETIC ACID proteins. Crl1 contains two putative auxin response elements (AuxREs) in its promoter region. The proximal AuxRE specifically interacted with a rice AUXIN RESPONSE FACTOR (ARF) and acted as a cis-motif for Crl1 expression. We conclude that Crl1 encodes a positive regulator for crown and lateral root formation and that its expression is directly regulated by an ARF in the auxin signaling pathway.     

Comparative proteome analyses of maize (Zea mays L.) primary roots prior to lateral root initiation reveal differential protein expression in the lateral root initiation mutant rum1

The embryonically preformed primary root is the first root type of maize that emerges after germination. In this study the abundant soluble proteins of 2.5-day-old primary roots of wild-type and lateral root mutant rum1 seedlings were compared before the initiation of lateral roots. In CBB-stained 2-D gels, among 350 detected proteins 14 were identified as differentially accumulated (>twofold change; t-test: 95% significance) in wild-type versus rum1 primary roots. These proteins which were identified via ESI MS/MS are encoded by 12 different genes. Functionally, these proteins are involved in lignin biosynthesis, defense, and the citrate cycle. Nine of these genes were further analyzed at the RNA expression level. This study represents the first comparative proteomic analysis of maize primary roots prior to lateral root initiation and will contribute to a better understanding of the molecular basis of root development in cereals.     

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.     

Proteomic analysis of shoot-borne root initiation in maize (Zea mays L.)

Postembryonically formed shoot-borne roots make up the major backbone of the adult maize root stock. In this study the abundant soluble proteins of the first node (coleoptilar node) of wild-type and mutant rtcs seedlings, which do not initiate crown roots, were compared at two early stages of crown root formation. In Coomassie Bluestained 2-D gels, representing soluble proteins of coleoptilar nodes 5 and 10 days after germination, 146 and 203 proteins were detected, respectively. Five differentially accumulated proteins (> two-fold change; t-test: 95% significance) were identified in 5-day-old and 14 differentially accumulated proteins in 10-day-old coleoptilar nodes of wild-type versus rtcs. All 19 differentially accumulated proteins were identified via ESI MS/MS mass spectrometry. Five differentially accumulated proteins, including a regulatory G-protein and a putative auxin-binding protein, were further analyzed at the RNA expression level. These experiments confirmed differential gene expression and revealed subtle developmental regulation of these genes during early coleoptilar node development. This study represents the first proteomic analysis of shoot-borne root initiation in cereals and will contribute to a better understanding of the molecular basis of this developmental process unique to cereals.     

Mortal: A Mutant of White Clover Defective in Nodal Root Development

A monogenic dominant mutant of white clover (Trifolium repens L.), designated Mortal, which is defective in the formation of adventitious nodal roots, is described. Mortal plants grown at temperatures ranging from 10 to 25°C do not initiate nodal root primordium development. However, all other aspects of plant development are normal, including the formation of lateral roots and wound-induced adventitious roots. In some genetic backgrounds, the Mortal mutation has a temperature-sensitive conditional phenotype. Mortal plants shifted from growing conditions of 20 to 30°C for 2 to 3 d form nodal root meristems. However, new nodes that develop after plants are returned to 20°C exhibit the mutant phenotype. The capacity to form nodal roots on cuttings placed in water is also influenced by the genetic background of the Mortal mutation. Genetic analysis established that the physiological reversion of Mortal to nodal root formation is controlled by at least two separate dominant genetic loci, one for Nodal water response (Now) and one for Nodal temperature response (Not); the Now locus has a dominant epistatic interaction with the Not locus. The conditional nature of Mortal should provide opportunities for the identification of genetic and physiological mechanisms that influence the development of nodal roots.Whereas the basic structure of angiosperms is established during embryogenesis, most organs are formed by postembryonic development (Esau, 1977). Generally, all of the shoot structures (leaves, nodes, internodes, axillary shoot meristems, and flowers) are derived from the primary shoot apical meristem. However, adventitious shoot-borne roots are an exception because they develop endogenously from differentiated parenchyma cells close to the vascular tissues (Lovell and White, 1986).Little is known about the genes that control adventitious shoot-borne root morphogenesis, despite their importance for anchorage, nutrient acquisition, and water uptake from the soil in a wide range of plant species. One approach to understanding the genetic mechanisms that underlie adventitious root initiation and development is to identify and characterize mutants altered in the process. At present, few mutants with defects in adventitious root development are known (Schiefelbein and Benfey, 1991). There are mutants of tomato that produce few or no adventitious roots (Butler, 1954; Zobel, 1991) and mutants of maize that are defective in the formation of lateral seminal roots, crown roots, or both lateral seminal and crown roots (Jenkins, 1930; de Miranda, 1980; Hetz et al., 1996).The general unpredictability in the formation of secondary roots on shoots complicates the analysis of the genetic and molecular mechanisms controlling adventitious root development. This can be minimized by characterizing the genetic control of the formation of adventitious nodal root primordia. In some plant species, adventitious root primordia arise in a precise and ordered manner during node development. One such example is the nodal roots that form on the prostrate stolons of white clover (Trifolium repens L.; Thomas, 1987). In T. repens, the nodes of each stolon alternate in orientation so that successive nodes produce leaves and axillary buds on opposite sides of the stolon (Erith, 1924).The organization of nodes and nodal roots in white clover is illustrated in Figure ​Figure1.1. Root primordia are typically absent from the first four nodes of wild-type white clover stolons, and nodes bearing the first five leaf primordia are enclosed within the leaf sheath of the first visible node (Erith, 1924; Thomas, 1987). The first nodal root primordium is initiated below the axillary shoot bud of the fifth node, and a second primordium forms above the axillary shoot of the sixth node. In the seventh node, the lowermost of each pair of nodal root primordia matures into a root apical meristem that grows out through both the stolon epidermis and the stipular sheath to form a visible root, whereas development of the uppermost nodal root meristem is normally arrested such that it remains within the stipule. Further growth of the uppermost nodal root meristem usually occurs only in very moist conditions. Figure 1Stolon morphology of a wild-type white clover plant. A, Underside view of the apical portion of a stolon showing the nodes (N), leaf stipule (S), and petiole (P). B, Schematic representation of nodal development. SAM, Shoot apical meristem; LRP, lower ...White clover (2n = 4× = 32) is predominantly an obligate outcrossing species with disomic inheritance. Therefore, populations are a heterogeneous mixture of highly heterozygous individuals. This heterogeneity and the associated plasticity in environmental response complicates genetic analysis of some developmental traits in white clover. However, there are dominant self-compatible alleles of the gametophytic S locus system of sexual incompatibility, which can be used to self plants for the genetic analysis of traits (Williams, 1987).To determine the genetic control of adventitious root formation in white clover, we have identified and characterized a spontaneous mutant, designated Mortal, which is defective in nodal root primordium initiation. When grown at 20°C, Mortal plants lacked nodal root primordia but were normal in other aspects of shoot and root morphology. However, in some genetic backgrounds, Mortal was conditional, responding to either a temperature shift to 30°C or to the placing of stolon cuttings in water, by developing nodal roots. Here we describe Mortal and provide a genetic model for responses of the mutant to these temperature-shift and water treatments.