Combined in silico/in vivo analysis of mechanisms providing for root apical meristem self-organization and maintenance

Background and Aims

The root apical meristem (RAM) is the plant stem cell niche which provides for the formation and continuous development of the root. Auxin is the main regulator of RAM functioning, and auxin maxima coincide with the sites of RAM initiation and maintenance. Auxin gradients are formed due to local auxin biosynthesis and polar auxin transport. The PIN family of auxin transporters plays a critical role in polar auxin transport, and two mechanisms of auxin maximum formation in the RAM based on PIN-mediated auxin transport have been proposed to date: the reverse fountain and the reflected flow mechanisms.

Methods

The two mechanisms are combined here in in silico studies of auxin distribution in intact roots and roots cut into two pieces in the proximal meristem region. In parallel, corresponding experiments were performed in vivo using DR5::GFP Arabidopsis plants.

Key Results

The reverse fountain and the reflected flow mechanism naturally cooperate for RAM patterning and maintenance in intact root. Regeneration of the RAM in decapitated roots is provided by the reflected flow mechanism. In the excised root tips local auxin biosynthesis either alone or in cooperation with the reverse fountain enables RAM maintenance.

Conclusions

The efficiency of a dual-mechanism model in guiding biological experiments on RAM regeneration and maintenance is demonstrated. The model also allows estimation of the concentrations of auxin and PINs in root cells during development and under various treatments. The dual-mechanism model proposed here can be a powerful tool for the study of several different aspects of auxin function in root.     

Auxin transport in maize roots in response to localized nitrate supply

Background and Aims

Roots typically respond to localized nitrate by enhancing lateral-root growth. Polar auxin transport has important roles in lateral-root formation and growth; however, it is a matter of debate whether or how auxin plays a role in the localized response of lateral roots to nitrate.

Methods

Treating maize (Zea mays) in a split-root system, auxin levels were quantified directly and polar transport was assayed by the movement of [³H]IAA. The effects of exogenous auxin and polar auxin transport inhibitors were also examined.

Key Results

Auxin levels in roots decreased more in the nitrate-fed compartment than in the nitrate-free compartment and nitrate treatment appeared to inhibit shoot-to-root auxin transport. However, exogenous application of IAA only partially reduced the stimulatory effect of localized nitrate, and auxin level in the roots was similarly reduced by local applications of ammonium that did not stimulate lateral-root growth.

Conclusions

It is concluded that local applications of nitrate reduced shoot-to-root auxin transport and decreased auxin concentration in roots to a level more suitable for lateral-root growth. However, alteration of root auxin level alone is not sufficient to stimulate lateral-root growth.     

Auxin increases the hydrogen peroxide (H2O2) concentration in tomato (Solanum lycopersicum) root tips while inhibiting root growth

Background and Aims

The hormone auxin and reactive oxygen species (ROS) regulate root elongation, but the interactions between the two pathways are not well understood. The aim of this study was to investigate how auxin interacts with ROS in regulating root elongation in tomato, Solanum lycopersicum.

Methods

Wild-type and auxin-resistant mutant, diageotropica (dgt), of tomato (S. lycopersicum ‘Ailsa Craig’) were characterized in terms of root apical meristem and elongation zone histology, expression of the cell-cycle marker gene Sl-CycB1;1, accumulation of ROS, response to auxin and hydrogen peroxide (H₂O₂), and expression of ROS-related mRNAs.

Key Results

The dgt mutant exhibited histological defects in the root apical meristem and elongation zone and displayed a constitutively increased level of hydrogen peroxide (H₂O₂) in the root tip, part of which was detected in the apoplast. Treatments of wild-type with auxin increased the H₂O₂ concentration in the root tip in a dose-dependent manner. Auxin and H₂O₂ elicited similar inhibition of cell elongation while bringing forth differential responses in terms of meristem length and number of cells in the elongation zone. Auxin treatments affected the expression of mRNAs of ROS-scavenging enzymes and less significantly mRNAs related to antioxidant level. The dgt mutation resulted in resistance to both auxin and H₂O₂ and affected profoundly the expression of mRNAs related to antioxidant level.

Conclusions

The results indicate that auxin regulates the level of H₂O₂ in the root tip, so increasing the auxin level triggers accumulation of H₂O₂ leading to inhibition of root cell elongation and root growth. The dgt mutation affects this pathway by reducing the auxin responsiveness of tissues and by disrupting the H₂O₂ homeostasis in the root tip.     

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.     

Endogenous cytokinin in developing kiwifruit is implicated in maintaining fruit flesh chlorophyll levels

Background and Aims

Green kiwifruit (Actinidia deliciosa) retain high concentrations of chlorophyll in the fruit flesh, whereas in gold-fleshed kiwifruit (A. chinensis) chlorophyll is degraded to colourless catabolites during fruit development, leaving yellow carotenoids visible. The plant hormone group the cytokinins has been implicated in the delay of senescence, and so the aim of this work was to investigate the link between cytokinin levels in ripening fruit and chlorophyll de-greening.

Methods

The expression of genes related to cytokinin metabolism and signal transduction and the concentration of cytokinin metabolites were measured. The regulation of gene expression was assayed using transient activation of the promoter of STAY-GREEN2 (SGR2) by cytokinin response regulators.

Key Results

While the total amount of cytokinin increased in fruit of both species during maturation and ripening, a high level of expression of two cytokinin biosynthetic gene family members, adenylate isopentenyltransferases, was only detected in green kiwifruit fruit during ripening. Additionally, high levels of O-glucosylated cytokinins were detected only in green kiwifruit, as was the expression of the gene for zeatin O-glucosyltransferase, the enzyme responsible for glucosylating cytokinin into a storage form. Season to season variation in gene expression was seen, and some de-greening of the green kiwifruit fruit occurred in the second season, suggesting environmental effects on the chlorophyll degradation pathway. Two cytokinin-related response regulators, RRA17 and RRB120, showed activity against the promoter of kiwifruit SGR2.

Conclusions

The results show that in kiwifruit, levels of cytokinin increase markedly during fruit ripening, and that cytokinin metabolism is differentially regulated in the fruit of the green and gold species. However, the causal factor(s) associated with the maintenance or loss of chlorophyll in kiwifruit during ripening remains obscure.     

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.     

Involvement of nitric oxide and auxin in signal transduction of copper-induced morphological responses in Arabidopsis seedlings

Background and Aims

Plants are able to adapt to the environment dynamically through regulation of their growth and development. Excess copper (Cu²⁺), a toxic heavy metal, induces morphological alterations in plant organs; however, the underlying mechanisms are still unclear. With this in mind, the multiple signalling functions of nitric oxide (NO) in plant cells and its possible regulatory role and relationship with auxin were examined during Cu²⁺-induced morphological responses.

Methods

Endogenous auxin distribution was determined by microscopic observation of X-Gluc-stained DR5::GUS arabidopsis, and the levels of NO, superoxide and peroxynitrite were detected by fluorescence microscopy. As well as wild-type, NO-overproducer (nox1) and -deficient (nia1nia2 and nia1nia2noa1-2) arabidopsis plants were used.

Key Results

Cu²⁺ at a concentration of 50 µm resulted in a large reduction in cotyledon area and hypocotyl and primary root lengths, accompanied by an increase in auxin levels. In cotyledons, a low Cu²⁺ concentration promoted NO accumulation, which was arrested by nitric oxide synthase or nitrate reductase inhibitors. The 5-μm Cu²⁺-induced NO synthesis was not detectable in nia1nia2 or nia1nia2noa1-2 plants. In roots, Cu²⁺ caused a decrease of the NO level which was not associated with superoxide and peroxynitrite formation. Inhibition of auxin transport resulted in an increase in NO levels, while exogenous application of an NO donor reduced DR5::GUS expression. The elongation processes of nox1 were not sensitive to Cu²⁺, but NO-deficient plants showed diverse growth responses.

Conclusions

In plant organs, Cu²⁺ excess results in severe morphological responses during which the endogenous hormonal balance and signal transduction are affected. Auxin and NO negatively regulate each other''s level and NO intensifies the metal-induced cotyledon expansion, but mitigates elongation processes under Cu²⁺ exposure.     

Spatial and temporal profiles of cytokinin biosynthesis and accumulation in developing caryopses of maize

Background and Aims

Cytokinins are a major group of plant hormones and are associated with various developmental processes. Developing caryopses of maize have high levels of cytokinins, but little is known about their spatial and temporal distribution. The localization and quantification of cytokinins was investigated in maize (Zea mays) caryopsis from 0 to 28 d after pollination together with the expression and localization of isopentenyltransferase ZmIPT1 involved in cytokinin biosynthesis and ZmCNGT, the gene putatively involved in N9-glucosylation.

Methods

Biochemical, cellular and molecular approaches resolved the overall cytokinin profiles, and several gene expression assays were used for two critical genes to assess cytokinin cell-specific biosynthesis and conversion to the biologically inactive form. Cytokinins were immunolocalized for the first time in maize caryopses.

Key Results

During the period 0–28 d after pollination (DAP): (1) large quantities of cytokinins were detected in the maternal pedicel region relative to the filial tissues during the early stages after fertilization; (2) unpollinated ovules did not accumulate cytokinins; (3) the maternal nucellar region showed little or no cytokinin signal; (4) the highest cytokinin concentrations in filial endosperm and embryo were detected at 12 DAP, predominantly zeatin riboside and zeatin-9-glucoside, respectively; and (5) a strong cytokinin immuno-signal was detected in specific cell types in the pedicel, endosperm and embryo.

Conclusions

The cytokinins of developing maize caryopsis may originate from both local syntheses as well as by transport. High levels of fertilization-dependent cytokinins in the pedicel suggest filial control on metabolism in the maternal tissue; they may also trigger developmental programmed cell death in the pedicel.     

Auxin distribution is differentially affected by nitrate in roots of two rice cultivars differing in responsiveness to nitrogen

Background and Aims

Although ammonium (NH₄⁺) is the preferred form of nitrogen over nitrate (NO₃⁻) for rice (Oryza sativa), lateral root (LR) growth in roots is enhanced by partial NO₃⁻ nutrition (PNN). The roles of auxin distribution and polar transport in LR formation in response to localized NO₃⁻ availability are not known.

Methods

Time-course studies in a split-root experimental system were used to investigate LR development patterns, auxin distribution, polar auxin transport and expression of auxin transporter genes in LR zones in response to localized PNN in ‘Nanguang’ and ‘Elio’ rice cultivars, which show high and low responsiveness to NO₃⁻, respectively. Patterns of auxin distribution and the effects of polar auxin transport inhibitors were also examined in DR5::GUS transgenic plants.

Key Results

Initiation of LRs was enhanced by PNN after 7 d cultivation in ‘Nanguang’ but not in ‘Elio’. Auxin concentration in the roots of ‘Nanguang’ increased by approx. 24 % after 5 d cultivation with PNN compared with NH₄⁺ as the sole nitrogen source, but no difference was observed in ‘Elio’. More auxin flux into the LR zone in ‘Nanguang’ roots was observed in response to NO₃⁻ compared with NH₄⁺ treatment. A greater number of auxin influx and efflux transporter genes showed increased expression in the LR zone in response to PNN in ‘Nanguang’ than in ‘Elio’.

Conclusions

The results indicate that higher NO₃⁻ responsiveness is associated with greater auxin accumulation in the LR zone and is strongly related to a higher rate of LR initiation in the cultivar ‘Nanguang’.     

Sequential induction of auxin efflux and influx carriers regulates lateral root emergence

In Arabidopsis, lateral roots originate from pericycle cells deep within the primary root. New lateral root primordia (LRP) have to emerge through several overlaying tissues. Here, we report that auxin produced in new LRP is transported towards the outer tissues where it triggers cell separation by inducing both the auxin influx carrier LAX3 and cell‐wall enzymes. LAX3 is expressed in just two cell files overlaying new LRP. To understand how this striking pattern of LAX3 expression is regulated, we developed a mathematical model that captures the network regulating its expression and auxin transport within realistic three‐dimensional cell and tissue geometries. Our model revealed that, for the LAX3 spatial expression to be robust to natural variations in root tissue geometry, an efflux carrier is required—later identified to be PIN3. To prevent LAX3 from being transiently expressed in multiple cell files, PIN3 and LAX3 must be induced consecutively, which we later demonstrated to be the case. Our study exemplifies how mathematical models can be used to direct experiments to elucidate complex developmental processes.     

Secretome analysis reveals effector candidates associated with broad host range necrotrophy in the fungal plant pathogen Sclerotinia sclerotiorum

Background

The white mold fungus Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a remarkably broad host range. The interaction of necrotrophs with their hosts is more complex than initially thought, and still poorly understood.

Results

We combined bioinformatics approaches to determine the repertoire of S. sclerotiorum effector candidates and conducted detailed sequence and expression analyses on selected candidates. We identified 486 S. sclerotiorum secreted protein genes expressed in planta, many of which have no predicted enzymatic activity and may be involved in the interaction between the fungus and its hosts. We focused on those showing (i) protein domains and motifs found in known fungal effectors, (ii) signatures of positive selection, (iii) recent gene duplication, or (iv) being S. sclerotiorum-specific. We identified 78 effector candidates based on these properties. We analyzed the expression pattern of 16 representative effector candidate genes on four host plants and revealed diverse expression patterns.

Conclusions

These results reveal diverse predicted functions and expression patterns in the repertoire of S. sclerotiorum effector candidates. They will facilitate the functional analysis of fungal pathogenicity determinants and should prove useful in the search for plant quantitative disease resistance components active against the white mold.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-336) contains supplementary material, which is available to authorized users.     

Local regulation of auxin transport in root-apex transition zone mediates aluminium-induced Arabidopsis root-growth inhibition

Aluminium (Al) stress is a major limiting factor for worldwide crop production in acid soils. In Arabidopsis thaliana, the TAA1-dependent local auxin biosynthesis in the root-apex transition zone (TZ), the major perception site for Al toxicity, is crucial for the Al-induced root-growth inhibition, while the mechanism underlying Al-regulated auxin accumulation in the TZ is not fully understood. In the present study, the role of auxin transport in Al-induced local auxin accumulation in the TZ and root-growth inhibition was investigated. Our results showed that PIN-FORMED (PIN) proteins such as PIN1, PIN3, PIN4 and PIN7 and AUX1/LAX proteins such as AUX1, LAX1 and LAX2 were all ectopically up-regulated in the root-apex TZ in response to Al stress and coordinately regulated local auxin accumulation in the TZ and root-growth inhibition. The ectopic up-regulation of PIN1 in the TZ under Al stress was regulated by both ethylene and auxin, with auxin signalling acting downstream of ethylene. Al-induced PIN1 up-regulation and auxin accumulation in the root-apex TZ was also regulated by the calossin-like protein BIG. Together, our results provide insight into how Al stress induces local auxin accumulation in the TZ and root-growth inhibition through the local regulation of auxin transport.     

DAR2 acts as an important node connecting cytokinin,auxin, SHY2 and PLT1/2 in root meristem size control

Cytokinin and auxin antagonistically affect cell proliferation and differentiation and thus regulate root meristem size by influencing the abundance of SHORT HYPOCOTYL2 (SHY2/IAA3). SHY2 affects auxin distribution in the root meristem by repressing the auxin-inducible expression of PIN-FORMED (PIN) auxin transport genes. The PLETHORA (PLT1/2) genes influence root meristem growth by promoting stem cells and transit-amplifying cells. However, the factors connecting cytokinin, auxin, SHY2 and PLT1/2 are largely unknown. In a recent study, we have shown that the DA1-related protein 2 (DAR2) acts downstream of cytokinin and SHY2 but upstream of PLT1/2 to affect root meristem size. Here, we discuss the possible molecular mechanisms by which Arabidopsis DAR2 controls root meristem size.     

The AUX1 LAX family of auxin influx carriers is required for the establishment of embryonic root cell organization in Arabidopsis thaliana

Background and Aims

The root meristem of the Arabidopsis thaliana mature embryo is a highly organized structure in which individual cell shape and size must be regulated in co-ordination with the surrounding cells. The objective of this study was to determine the role of the AUX1 LAX family of auxin import carriers during the establishment of the embryonic root cell pattern.

Methods

The radicle apex of single and multiple aux1 lax mutant mature embryos was used to evaluate the effect of this gene family upon embryonic root organization and root cap size, cell number and cell size.

Key Results

It was demonstrated here that mutations within the AUX1 LAX family are associated with changes in cell pattern establishment in the embryonic quiescent centre and columella. aux1 lax mutants have a larger radicle root cap than the wild type and this is associated with a significant increase in the root-cap cell number, average cell size, or both. Extreme disorganization of the radicle apex was observed among quadruple aux1 lax1 lax2 lax3 mutant embryos, but not in single aux1 null or in lax1, lax2 and lax3 single mutants, indicating redundancy within the AUX1 LAX family.

Conclusions

It was determined that the AUX1 LAX family of auxin influx facilitators participates in the establishment of cell pattern within the apex of the embryonic root in a gene-redundant fashion. It was demonstrated that aux1 lax mutants are affected in cell proliferation and cell growth within the radicle tip. Thus AUX1 LAX auxin importers emerge as new players in morphogenetic processes involved in patterning during embryonic root formation.Key words: AUX1 LAX genes, auxin, Arabidopsis thaliana, embryogenesis, meristem, radicle development, cell pattern establishment     

ZIFL1.1 transporter modulates polar auxin transport by stabilizing membrane abundance of multiple PINs in Arabidopsis root tip

Cell-to-cell directional flow of the phytohormone auxin is primarily established by polar localization of the PIN auxin transporters, a process tightly regulated at multiple levels by auxin itself. We recently reported that, in the context of strong auxin flows, activity of the vacuolar ZIFL1.1 transporter is required for fine-tuning of polar auxin transport rates in the Arabidopsis root. In particular, ZIFL1.1 function protects plasma-membrane stability of the PIN2 carrier in epidermal root tip cells under conditions normally triggering PIN2 degradation. Here, we show that ZIFL1.1 activity at the root tip also promotes PIN1 plasma-membrane abundance in central cylinder cells, thus supporting the notion that ZIFL1.1 acts as a general positive modulator of polar auxin transport in roots.