Composite Cucurbita pepo plants with transgenic roots as a tool to study root development

Background and Aims

In most plant species, initiation of lateral root primordia occurs above the elongation zone. However, in cucurbits and some other species, lateral root primordia initiation and development takes place in the apical meristem of the parental root. Composite transgenic plants obtained by Agrobacterium rhizogenes-mediated transformation are known as a suitable model to study root development. The aim of the present study was to establish this transformation technique for squash.

Methods

The auxin-responsive promoter DR5 was cloned into the binary vectors pKGW-RR-MGW and pMDC162-GFP. Incorporation of 5-ethynyl-2′-deoxyuridine (EdU) was used to evaluate the presence of DNA-synthesizing cells in the hypocotyl of squash seedlings to find out whether they were suitable for infection. Two A. rhizogenes strains, R1000 and MSU440, were used. Roots containing the respective constructs were selected based on DsRED1 or green fluorescent protein (GFP) fluorescence, and DR5::Egfp-gusA or DR5::gusA insertion, respectively, was verified by PCR. Distribution of the response to auxin was visualized by GFP fluorescence or β-glucuronidase (GUS) activity staining and confirmed by immunolocalization of GFP and GUS proteins, respectively.

Key Results

Based on the distribution of EdU-labelled cells, it was determined that 6-day-old squash seedlings were suited for inoculation by A. rhizogenes since their root pericycle and the adjacent layers contain enough proliferating cells. Agrobacterium rhizogenes R1000 proved to be the most virulent strain on squash seedlings. Squash roots containing the respective constructs did not exhibit the hairy root phenotype and were morphologically and structurally similar to wild-type roots.

Conclusions

The auxin response pattern in the root apex of squash resembled that in arabidopsis roots. Composite squash plants obtained by A. rhizogenes-mediated transformation are a good tool for the investigation of root apical meristem development and root branching.     

Control of root architecture and nodulation by the LATD/NIP transporter

The Medicago truncatula LATD/NIP gene is essential for the development of lateral and primary root and nitrogen-fixing nodule meristems as well as for rhizobial invasion of nodules. LATD/NIP encodes a member of the NRT1(PTR1) nitrate and di-and tri-peptide transporter family, suggesting that its function is to transport one of these or another compound(s). Because latd/nip mutants can have their lateral and primary root defects rescued by ABA, ABA is a potential substrate for transport. LATD/NIP expression in the root meristem was demonstrated to be regulated by auxin, cytokinin and abscisic acid, but not by nitrate. LATD/NIP''s potential function and its role in coordinating root architecture and nodule formation are discussed.Key words: nodule development, lateral root development, root architecture, symbiotic nitrogen fixation, Medicago truncatula, NRT1(PTR) gene familyUnlike most other plants, legumes form two kinds of lateral root organs: lateral roots and nitrogen-fixing root nodules that form in conjunction with compatible symbiotic rhizobium bacteria. Although the morphology and function of these two root organs is distinct, both require the function of the LATD/NIP gene, indicating shared genetic components for these two developmental processes and providing support for a model in which legume nodules evolved from a lateral root blueprint. Both lateral roots and nodules initiate in previously differentiated root cells in response to environmental and developmental cues mediated by hormones. Interestingly, regulation of nodules and lateral roots by hormones is often opposite, allowing formation of one organ or another depending on the conditions.     

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

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

Regulation of root morphogenesis in arbuscular mycorrhizae: what role do fungal exudates,phosphate, sugars and hormones play in lateral root formation?

Background

Arbuscular mycorrhizae (AMs) form a widespread root–fungus symbiosis that improves plant phosphate (Pi) acquisition and modifies the physiology and development of host plants. Increased branching is recognized as a general feature of AM roots, and has been interpreted as a means of increasing suitable sites for colonization. Fungal exudates, which are involved in the dialogue between AM fungi and their host during the pre-colonization phase, play a well-documented role in lateral root (LR) formation. In addition, the increased Pi content of AM plants, in relation to Pi-starved controls, as well as changes in the delivery of carbohydrates to the roots and modulation of phytohormone concentration, transport and sensitivity, are probably involved in increasing root system branching.

Scope

This review discusses the possible causes of increased branching in AM plants. The differential root responses to Pi, sugars and hormones of potential AM host species are also highlighted and discussed in comparison with those of the non-host Arabidopsis thaliana.

Conclusions

Fungal exudates are probably the main compounds regulating AM root morphogenesis during the first colonization steps, while a complex network of interactions governs root development in established AMs. Colonization and high Pi act synergistically to increase root branching, and sugar transport towards the arbusculated cells may contribute to LR formation. In addition, AM colonization and high Pi generally increase auxin and cytokinin and decrease ethylene and strigolactone levels. With the exception of cytokinins, which seem to regulate mainly the root:shoot biomass ratio, these hormones play a leading role in governing root morphogenesis, with strigolactones and ethylene blocking LR formation in the non-colonized, Pi-starved plants, and auxin inducing them in colonized plants, or in plants grown under high Pi conditions.     

Chloroplast protein PLGG1 is involved in abscisic acid-regulated lateral root development and stomatal movement in Arabidopsis

The plant hormone abscisic acid (ABA) plays a crucial role in root architecture; however, the molecular mechanism of ABA-regulated lateral root (LR) growth is not well known. We screened an Arabidopsis thaliana mutant with LR growth that was sensitive to ABA from a T-DNA insertion mutant library, which was an allelic mutant of plgg1-1, termed plgg1-2. PLGG1 encodes a chloroplast protein that transports plastidic glycolate and glycerate. The length and number of LRs at the root-hypocotyl junction of plgg1-1 and plgg1-2 were significantly impaired under exogenous ABA treatment, and the transgenic plant complementary lines of plgg1-2 restored LR growth in response to ABA. In addition, we found that PLGG1 is involved in other major ABA responses, including ABA-inhibited seed germination, ABA-mediated stomatal movement, and drought tolerance. These findings open new perspectives on elucidating the mechanism of ABA response, and provide clues for analysing the functions of chloroplast proteins in regulating root growth.     

Single-plant,Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.     

Ontogenetic contingency of tolerance mechanisms in response to apical damage

Background and Aims

Plants are able to tolerate tissue loss through vigorous branching which is often triggered by release from apical dominance and activation of lateral meristems. However, damage-induced branching might not be a mere physiological outcome of released apical dominance, but an adaptive response to environmental signals, such as damage timing and intensity. Here, branching responses to both factors were examined in the annual plant Medicago truncatula.

Methods

Branching patterns and allocation to reproductive traits were examined in response to variable clipping intensities and timings in M. truncatula plants from two populations that vary in the onset of reproduction. Phenotypic selection analysis was used to evaluate the strength and direction of selection on branching under the damage treatments.

Key Results

Plants of both populations exhibited an ontogenetic shift in tolerance mechanisms: while early damage induced greater meristem activation, late damage elicited investment in late-determined traits, including mean pod and seed biomass, and supported greater germination rates. Severe damage mostly elicited simultaneous development of multiple-order lateral branches, but this response was limited to early damage. Selection analyses revealed positive directional selection on branching in plants under early- compared with late- or no-damage treatments.

Conclusions

The results demonstrate that damage-induced meristem activation is an adaptive response that could be modified according to the plant''s developmental stage, severity of tissue loss and their interaction, stressing the importance of considering these effects when studying plastic responses to apical damage.     

Local and Systemic Regulation of Plant Root System Architecture and Symbiotic Nodulation by a Receptor-Like Kinase

In plants, root system architecture is determined by the activity of root apical meristems, which control the root growth rate, and by the formation of lateral roots. In legumes, an additional root lateral organ can develop: the symbiotic nitrogen-fixing nodule. We identified in Medicago truncatula ten allelic mutants showing a compact root architecture phenotype (cra2) independent of any major shoot phenotype, and that consisted of shorter roots, an increased number of lateral roots, and a reduced number of nodules. The CRA2 gene encodes a Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that primarily negatively regulates lateral root formation and positively regulates symbiotic nodulation. Grafting experiments revealed that CRA2 acts through different pathways to regulate these lateral organs originating from the roots, locally controlling the lateral root development and nodule formation systemically from the shoots. The CRA2 LRR-RLK therefore integrates short- and long-distance regulations to control root system architecture under non-symbiotic and symbiotic conditions.     

TTG1-mediated flavonols biosynthesis alleviates root growth inhibition in response to ABA

Key message

Our results demonstrate that the flavonoids biosynthetic pathway can be effectively manipulated to confer enhanced plant root growth under water-stress conditions.

Abstract

Abscisic acid (ABA) is one of most important phytohormones. It functions in various processes during the plant lifecycle. Previous studies indicate that ABA has a negative effect on root growth and branching. Auxin is another key plant growth regulator that plays an essential role in plant growth and development. In contrast to ABA, auxin is a positive regulator of root growth and development at low concentrations. This study was performed to help understand whether flavonoids can suppress the effect of ABA on lateral root growth. The recessive TRANSPARENT TESTA GLABRA 1 (ttg1) mutant was characterized on ABA and sucrose treatments. It was determined that auxin mobilization could be altered by modifying flavonoids biosynthesis, which resulted in alterations of root architecture in response to ABA treatment. Moreover, transgenic TTG1-overexpression (TTG1-OX) seedlings exhibited enhanced root length and lateral root number compared to wild-type seedlings grown under normal or stress conditions. Genetic manipulation of the flavonoids biosynthetic pathway could therefore be employed successfully for the improvement of plant root systems by overcoming the inhibition of ABA and some abiotic stresses.     

The CRE1 Cytokinin Pathway Is Differentially Recruited Depending on Medicago truncatula Root Environments and Negatively Regulates Resistance to a Pathogen

Cytokinins are phytohormones that regulate many developmental and environmental responses. The Medicago truncatula cytokinin receptor MtCRE1 (Cytokinin Response 1) is required for the nitrogen-fixing symbiosis with rhizobia. As several cytokinin signaling genes are modulated in roots depending on different biotic and abiotic conditions, we assessed potential involvement of this pathway in various root environmental responses. Phenotyping of cre1 mutant roots infected by the Gigaspora margarita arbuscular mycorrhizal (AM) symbiotic fungus, the Aphanomyces euteiches root oomycete, or subjected to an abiotic stress (salt), were carried out. Detailed histological analysis and quantification of cre1 mycorrhized roots did not reveal any detrimental phenotype, suggesting that MtCRE1 does not belong to the ancestral common symbiotic pathway shared by rhizobial and AM symbioses. cre1 mutants formed an increased number of emerged lateral roots compared to wild-type plants, a phenotype which was also observed under non-stressed conditions. In response to A. euteiches, cre1 mutants showed reduced disease symptoms and an increased plant survival rate, correlated to an enhanced formation of lateral roots, a feature previously linked to Aphanomyces resistance. Overall, we showed that the cytokinin CRE1 pathway is not only required for symbiotic nodule organogenesis but also affects both root development and resistance to abiotic and biotic environmental stresses.     

Conservation and divergence of signalling pathways between roots and soil microbes – the Rhizobium-legume symbiosis compared to the development of lateral roots, mycorrhizal interactions and nematode-induced galls

This review compares endophytic symbiotic and pathogenic root–microbe interactions and examines how the development of root structures elicited by various micro-organisms could have evolved by recruitment of existing plant developmental pathways. Plants are exposed to a multitude of soil micro-organisms which affect root development and performance. Their interactions can be of symbiotic and pathogenic nature, both of which can result in the formation of new root structures – how does the plant regulate the different outcomes of interactions with microbes? The idea that pathways activated in plant by micro-organisms could have been `hijacked' from plant developmental pathways is not new, it was essentially proposed by P. S. Nutman in 1948, but at that time, the molecular evidence to support that hypothesis was missing. Genetic evidence for overlaps between different plant–microbe interactions have previously been examined. This review compares the physiological and molecular plant responses to symbiotic rhizobia with those to arbuscular mycorrhizal fungi, pathogenic nematodes and the development of lateral roots and summarises evidence from both molecular and cellular studies for substantial overlaps in the signalling pathways underlying root–micro-organism interactions. A more difficult question has been why plant responses to micro-organisms are so similar, even though the outcomes are very different. Possible hypotheses for divergence of signalling pathways and future approaches to test these ideas are presented.     

A novel role for abscisic acid emerges from underground

The continuous formation of lateral roots is a vital part of establishing a root system and enables plants to react with developmental plasticity to changing soil conditions. Evidence is accumulating that abscisic acid (ABA), which is known to be involved in stress responses, has an important role in lateral root formation. An ABA receptor mutant, fca-1, shows an altered response to ABA during lateral root formation. Interestingly, ABA seems to have distinct roles at different stages in lateral root development. The emerging role of ABA in lateral root development fits well with its general functional properties as a stress hormone, including its role in dormancy.     

Nitric oxide is required for the auxin-induced activation of NADPH-dependent thioredoxin reductase and protein denitrosylation during root growth responses in arabidopsis

Background and Aims Auxin is the main phytohormone controlling root development in plants. This study uses pharmacological and genetic approaches to examine the role of auxin and nitric oxide (NO) in the activation of NADPH-dependent thioredoxin reductase (NTR), and the effect that this activity has on root growth responses in Arabidopsis thaliana.Methods Arabidopsis seedlings were treated with auxin with or without the NTR inhibitors auranofin (ANF) and 1-chloro-2, 4-dinitrobenzene (DNCB). NTR activity, lateral root (LR) formation and S-nitrosothiol content were measured in roots. Protein S-nitrosylation was analysed by the biotin switch method in wild-type arabidopsis and in the double mutant ntra ntrb.Key Results The auxin-mediated induction of NTR activity is inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), suggesting that NO is downstream of auxin in this regulatory pathway. The NTR inhibitors ANF and DNCB prevent auxin-mediated activation of NTR and LR formation. Moreover, ANF and DNCB also inhibit auxin-induced DR5 : : GUS and BA3 : : GUS gene expression, suggesting that the auxin signalling pathway is compromised without full NTR activity. Treatment of roots with ANF and DNCB increases total nitrosothiols (SNO) content and protein S-nitrosylation, suggesting a role of the NTR-thioredoxin (Trx)-redox system in protein denitrosylation. In agreement with these results, the level of S-nitrosylated proteins is increased in the arabidopsis double mutant ntra ntrb as compared with the wild-type.Conclusions The results support for the idea that NTR is involved in protein denitrosylation during auxin-mediated root development. The fact that a high NO concentration induces NTR activity suggests that a feedback mechanism to control massive and unregulated protein S-nitrosylation could be operating in plant cells.     

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.     

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.