The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer,cytological staining and 31P NMR spectroscopy study

Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP) synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non‐host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used ³²P labelling to quantify P accumulation and P efflux and in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long‐chain polyP in H. cylindrosporum if previously grown under P‐deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non‐host plants. However, the host plant enhanced ³²P release compared with the non‐host plant and specifically increased the proportion of short‐chain polyP in the interacting mycelia. These results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.     

Influence of auxin and mycorrhizal fungi on thein vitro formation and growth ofPinus pinaster roots

Summary Experiments, performed withPinus pinaster cloned shoots submitted to an auxin treatment (NAA 10^–6 M, 18 days), demonstrated that rooting abilityin vitro persists over 5 successive induction cycles (through out a 9-month period). Rooting ability needs a permanent synthesis of auxin synergists which activate the metabolism of cell dedifferentiation and root primordium initiation. Agar culture permitted intense meristem initiation, but prevented active root elongation. In the presence of a mycorrhizal fungus,Pisolithus tinctorius orHebeloma cylindrosporum, roots resumed growth and short lateral root formation was stimulated. These two phenomena induced by fungal association improve the quality of the root systems required to facilitate successful transplantation from test-tubes to field conditions.     

Local measurements of nitrate and potassium fluxes along roots of maritime pine. Effects of ectomycorrhizal symbiosis

In order to assess the actual role of ectomycorrhizae in ion uptake by the ectomycorrhizal root system, we used a microelectrode ion flux estimation methodology that provided access to local values of net fluxes. This made it possible to investigate the heterogeneity of ion fluxes along the different types of roots of Pinus pinaster associated or not with ectomycorrhizal species. We compared two fungi able to grow with nitrate in pure culture, Rhizopogon roseolus and Hebeloma cylindrosporum, the former having a positive effect on host tree shoot growth (c. +30%) and the latter a negative effect (c.? 30%). In non‐mycorrhizal plants (control), NO₃^– was taken up at higher rates by the short roots than by the long ones, whereas K⁺ uptake occurred mainly in growing apices of long roots. In mycorrhizal plants, H. cylindrosporum did not modify K⁺ uptake and even decreased NO₃^– uptake at the level of ectomycorrhizal short roots, whereas R. roseolus strongly increased K⁺ and NO₃^– fluxes at the level of ectomycorrhizal short roots without any modification of the fluxes measured along the fungus‐free long roots. The measurement of ion influxes at the surface of the ectomycorrhizal roots can provide a way to reveal actual effects of mycorrhizal association on ion transport in relation to mycorrhizal efficiency in natural conditions.     

Molecular Characterization of an Aux/IAA of Catharanthus roseus

In Catharanthus roseus cells, auxins are known to negatively regulate the biosynthesis of monoterpenoid indole alkaloids (MIA), a class of valuable secondary metabolites. Despite extensive studies of this regulation, no protein of the auxin signaling pathway has been isolated to date in this plant. We therefore decided to clone and characterize a C. roseus Aux/IAA protein that belongs to a family of gene expression repressors mediating auxin effects. Using PCR, a cDNA encoding the first C. roseus Aux/IAA was cloned and named CrIAA1. The deduced amino acid sequence has four highly conserved domains that are typical of the Aux/IAA protein family and has high homology to the Aux/IAA isoforms of Arabidopsis (>67%). The CrIAA1 gene expression, monitored by real-time PCR, was found to be dramatically induced by auxin treatment in C. roseus cells. Using GFP imagery and a bimolecular fluorescence complementation assay, we found that CrIAA1 can form oligomers in the nucleus. We also found that CrIAA1 is quickly degraded following auxin treatments, suggesting that auxin regulates CrIAA1 availability via a feedback mechanism. These results should help to elucidate the molecular nature of the processes responsible for the auxin-mediated regulation of MIA biosynthesis in C. roseus.     

Effect of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of arbuscular mycorrhiza-forming Prunus avium and Prunus cerasus

 The effect of different genotypes of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of Prunus avium and P. cerasus was studied in an attempt to determine whether ectomycorrhizal fungi could enhance in vitro adventitious root formation in plants which form arbuscular endomycorrhizas. The rooting percentage of P. avium cuttings was approximately 16% in the absence of hormonal treatment; it increased up to 30% in the presence of 5.7 μM IAA which was the most favourable auxin concentration. The rooting percentage of cuttings cultivated in the absence of IAA was enhanced by all the studied strains of H. cylindrosporum. It ranged from 50 to 60% with the IAA-overproducing mutant D 111 or the wild-type dikaryon D1, to 100% in the presence of the mutants 331 or D 117. The cuttings of P. cerasus showed a higher rooting ability than those of P. avium since approximately 40% of them were able to root in the absence of hormonal treatment. Except for the mutant D117, their rooting percentage was not significantly improved by H. cylindrosporum. Fungal inoculation also affected the survival of cuttings at acclimatization: 50% of the uninoculated P. avium cuttings survived whereas the survival percentage of inoculated cuttings ranged from 30 to 100% depending on the fungal genotype. With P. cerasus, the percentage of survival of uninoculated cuttings ranged from 85 to 100% and fungi either did not significantly improve it or lowered it. At acclimatization fungal hyphae could be observed in close contact with adventitious roots, but they did not establish mycorrhizal association. The shoot height of P. avium plantlets obtained from inoculated cuttings was not significantly different from that of plantlets originating from uninoculated ones. By contrast, fungal inoculation generally depressed the growth of acclimatized P. cerasus plantlets. The possibility of using ectomycorrhizal fungi as a tool to enhance rooting of micropropagated cuttings of plants which do not form ectomycorrhizas is discussed. Received: 25 November 1996 / Accepted: 2 June 1997     

外源色氨酸对油菜幼苗色氨酸下游代谢网络及生长发育的影响

色氨酸是合成蛋白质的重要氨基酸,也是植物生长激素IAA和某些次生代谢产物的前体物质,对植物生长发育及病虫害防御有重要作用。为了探究色氨酸对白菜型油菜(Brassica rapa L)生长发育及防御物质累积的影响及其可能的机制,该研究采用外源色氨酸对油菜幼苗进行叶面喷施,分析了色氨酸对油菜幼苗生长发育及生长素IAA和次生代谢产物芥子油苷合成的影响。结果表明：(1)低浓度色氨酸(100 mg/L)处理可有效地促进油菜叶片与根系的发育,但随着浓度增高,促进作用逐渐减弱。(2)荧光定量PCR分析表明,外源色氨酸处理后,油菜幼苗叶片中生长素IAA的3条合成途径都被激活,IPA途径的BrTAA1和BrYUCCA8、IAM途径的BrAMI1及IAOx途径的BrCYP71A13和BrNIT2等关键酶基因的表达均受到强烈的诱导,因而导致IAA的含量显著提高。(3)外源色氨酸处理还激活了下游的吲哚族芥子油苷的合成途径调控因子基因BrMYB34、BrMYB51和BrMYB122以及合成酶基因BrCYP79B2、BrCYP79B3、BrCYP83B1、BrSUR1的表达,同时抑制了其降解酶基因BrTGG1、BrPEN2的表达,从而引起吲哚族芥子油苷的累积。研究发现,外源色氨酸处理可通过调控生长素IAA合成途径和吲哚族芥子油苷的合成途径相关基因表达,有效地促进油菜生长调节物质和生物防御物质的累积,从而增加生物量和提高潜在抗病能力。     

Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum

Ectomycorrhizal fungi play an essential role in the ecology of boreal and temperate forests through the improvement of tree mineral nutrition. Potassium (K⁺) is an essential nutrient for plants and is needed in high amounts. We recently demonstrated that the ectomycorrhizal fungus Hebeloma cylindrosporum improves the K⁺ nutrition of Pinus pinaster under shortage conditions. Part of the transport systems involved in K⁺ uptake by the fungus has been deciphered, while the molecular players responsible for the transfer of this cation towards the plant remain totally unknown. Analysis of the genome of H. cylindrosporum revealed the presence of three putative tandem‐pore outward‐rectifying K⁺ (TOK) channels that could contribute to this transfer. Here, we report the functional characterization of these three channels through two‐electrode voltage‐clamp experiments in oocytes and yeast complementation assays. The expression pattern and physiological role of these channels were analysed in symbiotic interaction with P. pinaster. Pine seedlings colonized by fungal transformants overexpressing two of them displayed a larger accumulation of K⁺ in shoots. This study revealed that TOK channels have distinctive properties and functions in axenic and symbiotic conditions and suggested that HcTOK2.2 is implicated in the symbiotic transfer of K⁺ from the fungus towards the plant .     

IAA-overproducer mutants of Hebeloma cylindrosporum Romagnesi mycorrhizal with Pinus pinaster (Ait.) Sol. and P. sylvestris L. in hydroponic culture

Indole-3-acetic acid (IAA) is thought to play a role in the regulation of ectomycorrhiza development, and vigorous mycorrhiza formers such as Pisolithus and Laccaria have previously been shown to accumulate large amounts of IAA in the culture medium in vitro, particularly in the presence of tryptophan. Recently, 5-fluoroindole-resistant and IAA-overproducing mutant strains of Hebeloma cylindrosporum Romagnesi have been developed and described by Durand et al. (1992). We have used some of these and corresponding wild-type strains as mycobionts on seedlings of Pinus pinaster (Ait.) Sol. and P. sylvestris L. in semi-hydroponic culture in an attempt to study IAA effects independent of species-specific differences. However, no significant differences between strains were found in host growth rate, shoot carbohydrate concentration, root morphology, root IAA concentration or mycorrhizal biomass. Since previous work showed a stimulation by these and other mutants and strains on mycorrhiza formation in Petri dish and test tube cultures, we assume that a semi-hydroponic culture system prevents the build up of tryptophan of fungal origin, which is most likely a precondition for enhanced IAA production.     

The Interrelation of RNA, Auxin and Auxin-oxidases in Lentil Roots

The correlation between auxin and RNA metabolism was investigated in lentil roots. IAA and NAA both cause a considerable rise in the RNA level of germinating lentil roots, though no effect of IAA was found on the DNA level. In untreated germinating roots various sections were isolated and a direct relation found between RNA and auxin content, and an indirect relation between RNA content and auxin oxidase activity. In excised roots, incubated for 24 hours, the loss of RNA is paralleled by a loss of endogenous auxin. Excised roots treated with 10^?4M IAA or M 10^?4 NAA loose little RNA. The findings suggest that in lentil roots the RNA levels may be controlled by auxin levels, which in turn may be controlled by the levels of auxin oxidase.     

Small acidic protein 1 and SCFTIR1 ubiquitin proteasome pathway act in concert to induce 2,4‐dichlorophenoxyacetic acid‐mediated alteration of actin in Arabidopsis roots

2,4‐Dichlorophenoxyacetic acid (2,4‐D), a functional analogue of auxin, is used as an exogenous source of auxin as it evokes physiological responses like the endogenous auxin, indole‐3‐acetic acid (IAA). Previous molecular analyses of the auxin response pathway revealed that IAA and 2,4‐D share a common mode of action to elicit downstream physiological responses. However, recent findings with 2,4‐D‐specific mutants suggested that 2,4‐D and IAA might also use distinct pathways to modulate root growth in Arabidopsis. Using genetic and cellular approaches, we demonstrate that the distinct effects of 2,4‐D and IAA on actin filament organization partly dictate the differential responses of roots to these two auxin analogues. 2,4‐D but not IAA altered the actin structure in long‐term and short‐term assays. Analysis of the 2,4‐D‐specific mutant aar1‐1 revealed that small acidic protein 1 (SMAP1) functions positively to facilitate the 2,4‐D‐induced depolymerization of actin. The ubiquitin proteasome mutants tir1‐1 and axr1‐12, which show enhanced resistance to 2,4‐D compared with IAA for inhibition of root growth, were also found to have less disrupted actin filament networks after 2,4‐D exposure. Consistently, a chemical inhibitor of the ubiquitin proteasome pathway mitigated the disrupting effects of 2,4‐D on the organization of actin filaments. Roots of the double mutant aar1‐1 tir1‐1 also showed enhanced resistance to 2,4‐D‐induced inhibition of root growth and actin degradation compared with their respective parental lines. Collectively, these results suggest that the effects of 2,4‐D on actin filament organization and root growth are mediated through synergistic interactions between SMAP1 and SCF^TIR1 ubiquitin proteasome components.