Expression of MAX2 under SCARECROW promoter enhances the strigolactone/MAX2 dependent response of Arabidopsis roots to low-phosphate conditions.

MAIN CONCLUSION: MAX2/strigolactone signaling in the endodermis and/or quiescent center of the root is partially sufficient to exert changes in F-actin density and cellular trafficking in the root epidermis, and alter gene expression during plant response to low Pi conditions. Strigolactones (SLs) are a new group of plant hormones that regulate different developmental processes in the plant via MAX2, an F-box protein that interacts with their receptor. SLs and MAX2 are necessary for the marked increase in root-hair (RH) density in seedlings under conditions of phosphate (Pi) deprivation. This marked elevation was associated with an active reduction in actin-filament density and endosomal movement in root epidermal cells. Also, expression of MAX2 under the SCARECROW (SCR) promoter was sufficient to confer SL sensitivity in roots, suggesting that SL signaling pathways act through a root-specific, yet non-cell-autonomous regulatory mode of action. Here we show evidence for a non-cell autonomous signaling of SL/MAX2, originating from the root endodermis, and necessary for seedling response to conditions of Pi deprivation. SCR-derived expression of MAX2 in max2-1 mutant background promoted the root low Pi response, whereas supplementation of the synthetic SL GR24 to these SCR:MAX2 expressing lines further enhanced this response. Moreover, the SCR:MAX2 expression led to changes in actin density and endosome movement in epidermal cells and in TIR1 and PHO2 gene expression. These results demonstrate that MAX2 signaling in the endodermis and/or quiescent center is partially sufficient to exert changes in F-actin density and cellular trafficking in the epidermis, and alter gene expression under low Pi conditions.

Arabidopsis response to low-phosphate conditions includes active changes in actin filaments and PIN2 polarization and is dependent on strigolactone signalling.

Strigolactones (SLs) are plant hormones that regulate the plant response to phosphate (Pi) growth conditions. At least part of SL-signalling execution in roots involves MAX2-dependent effects on PIN2 polar localization in the plasma membrane (PM) and actin bundling and dynamics. We examined PIN2 expression, PIN2 PM localization, endosome trafficking, and actin bundling under low-Pi conditions: a MAX2-dependent reduction in PIN2 trafficking and polarization in the PM, reduced endosome trafficking, and increased actin-filament bundling were detected in root cells. The intracellular protein trafficking that is related to PIN proteins but unassociated with AUX1 PM localization was selectively inhibited. Exogenous supplementation of the synthetic SL GR24 to a SL-deficient mutant (max4) led to depletion of PIN2 from the PM under low-Pi conditions. Accordingly, roots of mutants in MAX2, MAX4, PIN2, TIR3, and ACTIN2 showed a reduced low-Pi response compared with the wild type, which could be restored by auxin (for all mutants) or GR24 (for all mutants except max2-1). Changes in PIN2 polarity, actin bundling, and vesicle trafficking may be involved in the response to low Pi in roots, dependent on SL/MAX2 signalling.

Strigolactone analog GR24 triggers changes in PIN2 polarity, vesicle trafficking and actin filament architecture.

Strigolactones (SLs) are plant hormones that regulate shoot and root development in a MAX2-dependent manner. The mechanism underlying SLs' effects on roots is unclear. We used root hair elongation to measure root response to SLs. We examined the effects of GR24 (a synthetic, biologically active SL analog) on localization of the auxin efflux transporter PIN2, endosomal trafficking, and F-actin architecture and dynamics in the plasma membrane (PM) of epidermal cells of the primary root elongation zone in wildtype (WT) Arabidopsis and the SL-insensitive mutant max2. We also recorded the response to GR24 of trafficking (tir3), actin (der1) and PIN2 (eir1) mutants. GR24 increased polar localization of PIN2 in the PM of epidermal cells and accumulation of PIN2-containing brefeldin A (BFA) bodies, increased ARA7-labeled endosomal trafficking, reduced F-actin bundling and enhanced actin dynamics, all in a MAX2-dependent manner. Most of the der1 and tir3 mutant lines also displayed reduced sensitivity to GR24 with respect to root hair elongation. We suggest that SLs increase PIN2 polar localization, PIN2 endocytosis, endosomal trafficking, actin debundling and actin dynamics in a MAX2-dependent fashion. This enhancement might underlie the WT root's response to SLs, and suggests noncell autonomous activity of SLs in roots.

Strigolactones are involved in root response to low phosphate conditions in Arabidopsis.

Strigolactones (SLs) are plant hormones that suppress lateral shoot branching, and act to regulate root hair elongation and lateral root formation. Here, we show that SLs are regulators of plant perception of or response to low inorganic phosphate (Pi) conditions. This regulation is mediated by MORE AXILLARY GROWTH2 (MAX2) and correlated with transcriptional induction of the auxin receptor TRANSPORT INHIBITOR RESPONSE1 (TIR1). Mutants of SL signaling (max2-1) or biosynthesis (max4-1) showed reduced response to low Pi conditions relative to the wild type. In max4-1, but not max2-1, the reduction in response to low Pi was compensated by the application of a synthetic strigolactone GR24. Moreover, AbamineSG, which decreases SL levels in plants, reduced the response to low Pi in the wild type, but not in SL-signaling or biosynthesis mutants. In accordance with the reduced response of max2-1 to low Pi relative to the wild type, several phosphate-starvation response and phosphate-transporter genes displayed reduced induction in max2-1, even though Pi content in max2-1 and the wild type were similar. Auxin, but not ethylene, was sufficient to compensate for the reduced max2-1 response to low Pi conditions. Moreover, the expression level of TIR1 was induced under low Pi conditions in the wild type, but not in max2-1. Accordingly, the tir1-1 mutant showed a transient reduction in root hair density in comparison with the wild type under low Pi conditions. Therefore, we suggest that the response of plants to low Pi is regulated by SLs; this regulation is transmitted via the MAX2 component of SL signaling and is correlated with transcriptional induction of the TIR1 auxin receptor.

Strigolactones affect lateral root formation and root-hair elongation in Arabidopsis.

Strigolactones (SLs) have been proposed as a new group of plant hormones, inhibiting shoot branching, and as signaling molecules for plant interactions. Here, we present evidence for effects of SLs on root development. The analysis of mutants flawed in SLs synthesis or signaling suggested that the absence of SLs enhances lateral root formation. In accordance, roots grown in the presence of GR24, a synthetic bioactive SL, showed reduced number of lateral roots in WT and in max3-11 and max4-1 mutants, deficient in SL synthesis. The GR24-induced reduction in lateral roots was not apparent in the SL signaling mutant max2-1. Moreover, GR24 led to increased root-hair length in WT and in max3-11 and max4-1 mutants, but not in max2-1. SLs effect on lateral root formation and root-hair elongation may suggest a role for SLs in the regulation of root development; perhaps, as a response to growth conditions.

Strigolactones interact with ethylene and auxin in regulating root-hair elongation in Arabidopsis.

Strigolactones (SLs) or derivatives thereof have been identified as phytohormones, and shown to act as long-distance shoot-branching inhibitors. In Arabidopsis roots, SLs have been suggested to have a positive effect on root-hair (RH) elongation, mediated via the MAX2 F-box. Two other phytohormones, auxin and ethylene, have been shown to have positive effects on RH elongation. Hence, in the present work, Arabidopsis RH elongation was used as a bioassay to determine epistatic relations between SLs, auxin, and ethylene. Analysis of the effect of hormonal treatments on RH elongation in the wild type and hormone-signalling mutants suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs, whereas the effect of SLs on RH elongation requires ethylene synthesis. SL signalling was not needed for the auxin response, whereas auxin signalling was not necessary, but enhanced RH response to SLs, suggesting that the SL and auxin hormonal pathways converge for regulation of RH elongation. The ethylene pathway requirement for the RH response to SLs suggests that ethylene forms a cross-talk junction between the SL and auxin pathways.

Strigolactone deficiency confers resistance in tomato line SL-ORT1 to the parasitic weeds Phelipanche and Orobanche spp.

The parasitic flowering plants of the genera Orobanche and Phelipanche (broomrape species) are obligatory chlorophyll-lacking root-parasitic weeds that infect dicotyledonous plants and cause heavy economic losses in a wide variety of plant species in warm-temperate and subtropical regions. One of the most effective strategies for broomrape control is crop breeding for broomrape resistance. Previous efforts to find natural broomrape-resistant tomato (Solanum lycopersicon) genotypes were unsuccessful, and no broomrape resistance was found in any wild tomato species. Recently, however, the fast-neutron-mutagenized tomato mutant SL-ORT1 was found to be highly resistant to various Phelipanche and Orobanche spp. Nevertheless, SL-ORT1 plants were parasitized by Phelipanche aegyptiaca if grown in pots together with the susceptible tomato cv. M-82. In the present study, no toxic activity or inhibition of Phelipanche seed germination could be detected in the SL-ORT1 root extracts. SL-ORT1 roots did not induce Phelipanche seed germination in pots but they were parasitized, at the same level as M-82, after application of the synthetic germination stimulant GR24 to the rhizosphere. Whereas liquid chromatography coupled to tandem mass spectrometry analysis of root exudates of M-82 revealed the presence of the strigolactones orobanchol, solanacol, and didehydro-orobanchol isomer, these compounds were not found in the exudates of SL-ORT1. It can be concluded that SL-ORT1 resistance results from its inability to produce and secrete natural germination stimulants to the rhizosphere.

Strigolactones are positive regulators of light-harvesting genes in tomato.

Strigolactones are newly identified plant hormones, shown to participate in the regulation of lateral shoot branching and root development. However, little is known about their effects on biological processes, genes, and proteins. Transcription profiling of roots treated with GR24, a synthetic strigolactone with proven biological activity, and/or indole acetic acid (IAA) was combined with physiological and transcriptional analysis of a tomato mutant (Sl-ORT1) deficient in strigolactone production. GR24 treatment led to markedly induced expression of genes putatively involved in light harvesting. This was apparent in both the presence and absence of exogenously applied IAA, but not with IAA treatment alone. Following validation of the microarray results, transcriptional induction by light of the GR24-induced genes was demonstrated in leaves exposed to high or low light intensities. Sl-ORT1 contained less chlorophyll and showed reduced expression of light harvesting-associated genes than the wild type (WT). Moreover, perfusion of GR24 into WT and Sl-ORT1 leaves led to induction of most of the examined light harvesting-associated genes. Results suggest that GR24 treatment interferes with the root's response to IAA treatment and that strigolactones are potentially positive regulators of light harvesting in plants.

A tomato strigolactone-impaired mutant displays aberrant shoot morphology and plant interactions.

Strigolactones are considered a new group of plant hormones. Their role as modulators of plant growth and signalling molecules for plant interactions first became evident in Arabidopsis, pea, and rice mutants that were flawed in strigolactone production, release, or perception. The first evidence in tomato (Solanum lycopersicon) of strigolactone deficiency is presented here. Sl-ORT1, previously identified as resistant to the parasitic plant Orobanche, had lower levels of arbuscular mycorrhizal fungus (Glomus intraradices) colonization, possibly as a result of its reduced ability to induce mycorrhizal hyphal branching. Biochemical analysis of mutant root extracts suggested that it produces only minute amounts of two of the tomato strigolactones: solanacol and didehydro-orobanchol. Accordingly, the transcription level of a key enzyme (CCD7) putatively involved in strigolactone synthesis in tomato was reduced in Sl-ORT1 compared with the wild type (WT). Sl-ORT1 shoots exhibited increased lateral shoot branching, whereas exogenous application of the synthetic strigolactone GR24 to the mutant restored the WT phenotype by reducing the number of lateral branches. Reduced lateral shoot branching was also evident in grafted plants which included a WT interstock, which was grafted between the mutant rootstock and the scion. In roots of these grafted plants, the CCD7 transcription level was not significantly induced, nor was mycorrhizal sensitivity restored. Hence, WT-interstock grafting, which restores mutant shoot morphology to WT, does not restore mutant root properties to WT. Characterization of the first tomato strigolactone-deficient mutant supports the putative general role of strigolactones as messengers of suppression of lateral shoot branching in a diversity of plant species.

Isolation of a premycorrhizal infection (pmi2) mutant of tomato, resistant to arbuscular mycorrhizal fungal colonization.

Arbuscular mycorrhizae (AM) represent an ancient symbiosis between mycorrhizal fungi and plant roots which co-evolved to exhibit a finely tuned, multistage interaction that assists plant growth. Direct screening efforts for Myc- plant mutants resulted in the identification of a tomato (Lycopersicon esculentum L. cv. Micro-Tom) mutant, M20, which was impaired in its ability to support the premycorrhizal infection (pmi) stages. The Myc- phenotype of the M20 mutant was a single Mendelian recessive trait, stable for nine generations, and nonallelic to a previously identified M161 pmi mutant. The M20 mutant was resistant to infection by isolated AM spores and colonized roots. Formation of Glomus intraradices appressoria on M20 roots was normal, as on wild-type (WT) plants, but in significantly reduced numbers. A significant reduction in spore germination was observed in vitro in the presence of M20 exudates relative to WT. Our results indicate that this new mutant shares similar physiological characteristics with the M161 pmi mutant, but has a more suppressive Myc- phenotype response.

Hormone concentrations in tobacco roots change during arbuscular mycorrhizal colonization with Glomus intraradices.

• Phytohormones are known to play a pivotal role in various developmental processes in plants and in arbuscular-mycorrhizal (AM) fungal-host symbiosis. This study focuses on characterizing the changes in the concentrations of auxins, cytokinins and gibberellins in tobacco (Nicotiana tabacum) during the early stages of colonization by Glomus intraradices, using advanced analytical detection techniques. • High-pressure liquid chromatography analysis followed by radioimmunoassay detection revealed that AM colonization induced the accumulation of specific zeatin riboside-like and isopentenyl adenosine-like compounds in both roots and shoots. • Use of the gas chromatography-mass spectrometry technique on the same developmental stage revealed that gibberellins (GA) of the earl-13-hydroxylation biosynthetic pathway (GA1 , GA8 , GA19 and GA20 ) were significantly more abundant in roots, but not shoots, of AM inoculated plants than in those of nonmycorrhizal plants. Indoleacetic acid concentrations (total and free) remained unaltered by AM colonization. • This study demonstrates that hormonal changes do occur during AM symbiosis with tobacco, before the fungal benefits manifest.

Root exudate of pmi tomato mutant M161 reduces AM fungal proliferation in vitro.

Soluble factors released from roots of the pre-mycorrhizal infection (pmi) myc(-) tomato mutant M161 were analyzed and compared with normal wild-type released factors. Aseptic whole exudates from the M161 mutant retarded the proliferation of Glomus intraradices in vitro. When the whole exudate was further fractionated on a C18 SEPAK cartridge, the 50/70% methanol fraction showed an activity against hyphal tip growth of Gigaspora gigantea and Gl. intraradices. Preliminary characterization of the exudate suggests that the inhibitory moieties are heat labile, bind to PVPP (polyvinyl polypyrrolidone), and are not volatile. This is the first reported instance of the inhibition by a myc(-) plant being ascribed to inhibitory component(s) released in root exudate.

Expression of streptavidin in tomato resulted in abnormal plant development that could be restored by biotin application.

Biotin is an essential cofactor for a variety of carboxylase and decarboxylase reactions and is involved in diverse metabolic pathways of all organisms. In the present study we tested the hypothesis that controlling biotin availability by the expression of Streptomyces avidinii streptavidin, would impede plant development. Transient expression of streptavidin fused to plant signal peptide, bacterial signal peptide or both, in tomato (Lycopersicon esculentum cv. VF36) plants resulted in various levels of tissue impairment, exhibited as lesion development on 1-week-old tomato seedlings. The least toxic construct was introduced to tomato (stable transformation) under the constitutive CaMV 35S promoter, and lesions appeared on stems, flower morphologies were modified and numbers and sizes of fruits were altered. Furthermore, tissue-specific expression of the streptavidin, by means of the beta-phaseolin or TobRB7 promoters, resulted in localised effects, i.e., impaired seed formation or seedless fruits, respectively, with no alteration in the morphology of the other plant organs. External application of biotin on streptavidin-expressing tomato plants prevented the degeneration symptoms and facilitated normal plant development. It can be concluded that expression of streptavidin in the plant cell can lead to local and temporal deficiencies in biotin availability, impairing developmental processes while biotin application restores plant growth cycle.

Structure-function relations of strigolactone analogs: activity as plant hormones and plant interactions.

Strigolactones (SLs) have several functions as signaling molecules in their interactions with symbiotic arbuscular mycorrhizal (AM) fungi and the parasitic weeds Orobanche and Striga. SLs are also a new class of plant hormone regulating plant development. In all three organisms, a specific and sensitive receptor-mediated perception system is suggested. By comparing the activity of synthetic SL analogs on Arabidopsis root-hair elongation, Orobanche aegyptiaca seed germination, and hyphal branching of the AM fungus Glomus intraradices, we found that each of the tested organisms differs in its response to the various examined synthetic SL analogs. Structure-function relations of the SL analogs suggest substitutions on the A-ring as the cause of this variation. Moreover, the description of competitive antagonistic analogs suggests that the A-ring of SL can affect not only affinity to the receptor, but also the molecule's ability to activate it. The results support the conclusion that Arabidopsis, Orobanche, and AM fungi possess variations in receptor sensitivity to SL analogs, probably due to variation in SL receptors among the different species.

Light is a positive regulator of strigolactone levels in tomato roots.

Strigolactones (SLs) or closely related molecules were recently identified as phytohormones, acting as long-distance branching factors that suppress growth of pre-formed axillary buds in the shoot. The SL signaling pathways and light appear to be connected, as SLs were shown to induce light-regulated pathways and to mimic light-adapted plant growth. However, it is not yet clear how light affects SL levels. Here, we examined the effect of different light intensities on SL levels in tomato roots. The results show that light intensity, above a certain threshold, is a positive regulator of SL levels and of Sl-CCD7 transcription; Sl-CCD7 is involved in SLs biosynthesis in tomato. Moreover, SL accumulation in plant roots is shown to be a time-dependent process. At least some of the similar effects of light and SLs on plant responses might result from a positive effect of light on SL levels.

Microarray analysis and functional tests suggest the involvement of expansins in the early stages of symbiosis of the arbuscular mycorrhizal fungus Glomus intraradices on tomato (Solanum lycopersicum).

Arbuscular mycorrhizal (AM) symbiosis occurs between fungi of the phylum Glomeromycota and most terrestrial plants. However, little is known about the molecular symbiotic signalling between AM fungi (AMFs) and non-leguminous plant species. We sought to further elucidate the molecular events occurring in tomato, a non-leguminous host plant, during the early, pre-symbiotic stage of AM symbiosis, i.e. immediately before and after contact between the AMF (Glomus intraradices) and the host. We adopted a semi-synchronized AMF root infection protocol, followed by genomic-scale, microarray-based, gene expression profiling at several defined time points during pre-symbiotic AM stages. The microarray results suggested differences in the number of differentially expressed genes and in the differential regulation of several functional groups of genes at the different time points examined. The microarray results were validated and one of the genes induced during contact between AMF and tomato, the expansin-like EXLB1, was functionally analysed. Expansins, encoded by a large multigene family, facilitate plant cell expansion. However, no biological or biochemical function has yet been established for plant-originated expansin-like proteins. EXLB1 transcripts were localized early during the association to cells that may perceive the fungal signal, and later during the association in close proximity to sites of AMF hypha-root colonization. Moreover, in transgenic roots, we demonstrated that a reduction in the steady-state level of EXLB1 transcript was correlated with a reduced rate of infection, reduced arbuscule expansion and reduced AMF spore formation.

Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress.

Most of the symplastic water transport in plants occurs via aquaporins, but the extent to which aquaporins contribute to plant water status under favorable growth conditions and abiotic stress is not clear. To address this issue, we constitutively overexpressed the Arabidopsis plasma membrane aquaporin, PIP1b, in transgenic tobacco plants. Under favorable growth conditions, PIP1b overexpression significantly increased plant growth rate, transpiration rate, stomatal density, and photosynthetic efficiency. By contrast, PIP1b overexpression had no beneficial effect under salt stress, whereas during drought stress it had a negative effect, causing faster wilting. Our results suggest that symplastic water transport via plasma membrane aquaporins represents a limiting factor for plant growth and vigor under favorable conditions and that even fully irrigated plants face limited water transportation. By contrast, enhanced symplastic water transport via plasma membrane aquaporins may not have any beneficial effect under salt stress, and it has a deleterious effect during drought stress.