The extraradical proteins of Rhizophagus irregularis: A shotgun proteomics approach

Arbuscular Mycorrhizal fungi (AMF, Glomeromycota) form obligate symbiotic associations with the roots of most terrestrial plants. Our understanding of the molecular mechanisms enabling AMF propagation and AMF-host interaction is currently incomplete. Analysis of AMF proteomes could yield important insights and generate hypotheses on the nature and mechanism of AMF-plant symbiosis. Here, we examined the extraradical mycelium proteomic profile of the arbuscular mycorrhizal fungus Rhizophagus irregularis grown on Ri T-DNA transformed Chicory roots in a root organ culture setting. Our analysis detected 529 different peptides that mapped to 474 translated proteins in the R. irregularis genome. R. irregularis proteome was characterized by a high proportion of proteins (9.9 % of total, 21.4 % of proteins with functional prediction) mediating a wide range of signal transduction processes, e.g. Rho1 and Bmh2, Ca-signaling (calmodulin, and Ca channel protein), mTOR signaling (MAP3K7, and MAPKAP1), and phosphatidate signaling (phospholipase D1/2) proteins, as well as members of the Ras signaling pathway. In addition, the proteome contained an unusually large proportion (53.6 %) of hypothetical proteins, the majority of which (85.8 %) were Glomeromycota-specific. Forty-eight proteins were predicted to be surface/membrane associated, including multiple hypothetical proteins of yet-unrecognized functions. However, no evidence for the overproduction of specific proteins, previously implicated in promoting soil health and aggregation was obtained. Finally, the comparison of R. irregularis proteome to previously published AMF proteomes identified a core set of pathways and processes involved in AMF growth. We conclude that R. irregularis growth on chicory roots requires the activation of a wide range of signal transduction pathways, the secretion of multiple novel hitherto unrecognized Glomeromycota-specific proteins, and the expression of a wide array of surface-membrane associated proteins for cross kingdom cell-to-cell communications.     

北方两省农牧交错带沙棘根围AM真菌与球囊霉素空间分布

2009年7月在内蒙古和河北两省农牧交错带沙棘(Hippophae rhamnoides L)集中分布区选取3个典型样地,分别从0-10cm、10-20cm、20-30cm、30-40cm和40-50cm土层采集沙棘根围土壤样品,研究了沙棘根围AM真菌与球囊霉素空间分布及与土壤因子的相关性。结果表明,沙棘能与AM真菌形成良好的疆南星型(Arum-type)丛枝菌根。AM真菌定殖率和孢子密度与样地生态条件密切相关。大梁底村和多伦东样地AM真菌不同结构定殖率及孢子密度无明显差别,但均显著高于黄柳条村样地,只在大梁底村发现丛枝定殖;孢子密度在3个样地均随土层加深而降低,不同结构定殖率在大梁底村随土层加深而降低,其他2个样地无明显变化规律;AM真菌最高定殖率和最大孢子密度均出现在0-30cm浅土层。根围土壤总球囊霉素(TEG)和易提取球囊霉素(EEG)含量在3个样地均随土层加深而降低。相关性分析表明,孢子密度与菌丝定殖率、泡囊定殖率和总定殖率极显著正相关。AM真菌菌丝、泡囊和总定殖率与土壤pH值、有机C、碱解N和速效P含量、脲酶和碱性磷酸酶活性极显著正相关,丛枝定殖率与土壤碱解N含量和脲酶活性显著正相关。多元线性回归方程表明,AM真菌不同定殖结构和土壤因子对TEG和EEG含量贡献不同,对于TEG:菌丝>孢子>泡囊;有机C>速效P>酸性磷酸酶>pH值,对于EEG:泡囊>孢子>菌丝;有机C>速效P。结果建议,AM真菌孢子密度、菌丝定殖程度和土壤球囊霉素含量在一定程度上能综合反应土壤AM真菌群落、有机C动态和养分循环进程,可以作为土壤质量及功能评价的新指标进一步深入研究。     

Improving soil protein extraction for metaproteome analysis and glomalin‐related soil protein detection

Soil contains low amounts of protein but high amounts of interfering substances. Current extraction methods for soil protein cannot produce high‐quality samples suitable for proteomic analysis. To resolve the problem, we devised a sequential extraction method, through sequentially extracting soil in citrate and SDS buffers, followed by phenol extraction. The method allows for obtaining applicable 1‐D and 2‐D protein profiles with various agricultural soils and detecting glomalin‐related soil protein. The method may be a valuable tool for soil proteomics.     

Bradford reactive soil protein in Appalachian soils: distribution and response to incubation, extraction reagent and tannins

Bradford reactive soil protein (BRSP) is thought to correlate to glomalin, an important soil glycoprotein that promotes soil aggregate formation and may represent a significant pool of stable soil organic matter (SOM). However, more information is needed about its importance in Appalachian soils and its relationships with other soil properties. We measured BRSP in 0–20 cm soil from pastures, hayfields, cultivated fields or forest areas in southern West Virginia. Highest amounts of BRSP were found near the soil surface and decreased significantly with depth for all land uses except cultivated sites. Forest and pasture sites contained more BRSP than hayfields or cultivated fields but these differences occurred only in the 0–5 cm depth. Overall averages of C and N in BRSP represented about 4.0 and 6.5% of the total soil C and N respectively. During a 395 day soil incubation, we found CO₂–C evolution rates comparable to other studies but only small changes in BRSP (<10%) including some evidence for increases during incubation. Sodium citrate, sodium pyrophosphate, and sodium oxalate recovered significantly more BRSP from soil than the other extractants we tested with highest extraction efficiencies observed for sodium citrate and pyrophosphate. Recovery of BRSP appears related to negative charge and buffering capacity of both the soil and extractant. Extractants with low negative charge had little buffering capacity and yielded little BRSP. Tannic acid appeared to increase extraction of BRSP but less soluble-N was recovered from tannin-treated samples than from untreated controls and E4/E6, the ratio of absorbance at 465 and 665 nm, decreased, evidence for the formation of larger or heavier molecules. Formation of dark-colored substances during extraction suggests the colorimetric Bradford assay may overestimate soil protein when tannins are present. Recovery of less soluble-N from soil extracts and lower E4/E6 ratios suggests tannins may bind with soil constituents themselves or form non-extractable N-containing complexes.     

Large contribution of arbuscular mycorrhizal fungi to soil carbon pools in tropical forest soils

The origins and composition of soil organic matter (SOM) are still largely uncertain. Arbuscular mycorrhizal fungi (AMF) are recognized as indirect contributors through their influence on soil aggregation, plant physiology, and plant community composition. Here we present evidence that AMF can also make large, direct contributions to SOM. Glomalin, a recently discovered glycoprotein produced by AMF hyphae, was detected in tropical soils in concentrations of over 60 mg cm^–3. Along a chronosequence of soils spanning ages from 300 to 4.1 Mio years, a pattern of glomalin concentrations is consistent with the hypothesis that this protein accumulates in soil. Carbon dating of glomalin indicated turnover at time scales of several years to decades, much longer than the turnover of AMF hyphae (which is assumed to be on the order of days to weeks). This suggests that contributions of mycorrhizae to soil carbon storage based on hyphal biomass in soil and roots may be an underestimate. The amount of C and N in glomalin represented a sizeable amount (ca. 4–5%) of total soil C and N in the oldest soils. Our results thus indicate that microbial (fungal) carbon that is not derived from above- or below-ground litter can make a significant contribution to soil carbon and nitrogen pools and can far exceed the contributions of soil microbial biomass (ranging from 0.08 to 0.2% of total C for the oldest soils).     

Responses of Bradford-reactive soil protein to experimental nitrogen addition in three forest communities in northern lower Michigan

Increased use of anthropogenically fixed N and the release of N in combustion products have led to concerns about possible long-term impacts on terrestrial ecosystems. Previous studies demonstrating the potential of atmospheric N deposition to influence forest soil carbon have focused on decomposition processes with much less known about potential impacts on mycorrhiza-derived carbon. Glomalin is a unique glycoprotein produced by arbuscular mycorrhizal (AM) fungi that has been implicated in the formation of soil aggregates and potentially a significant store of soil carbon. To determine the possible impact of experimental N deposition of such stores we examined the operationally defined glomalin-related soil protein (GRSP) levels over two growing seasons in three forest types receiving background N deposition (control) or treated with 80 kg N ha⁻¹ year⁻¹ as NaNO₃. Three sites of each of three forest types, sugar maple-basswood (SMBW), sugar maple-red oak (SMRO), and black oak-white oak (BOWO), in northern Lower Michigan were studied during the 2001 and 2002 growing seasons. GRSP were extracted from air-dried soils with citric acid and measured by the Bradford method. Analysis of variance revealed significant differences related to forest type and sample date in easily extractable Bradford reactive (EE-BRSP) and Bradford-reactive soil protein (BRSP), but failed to detect significant effects of experimental N amendment. EE-BRSP and BRSP varied in a pattern that was consistent with an AM fungal origin; a pattern that reflected the mycorrhizal types of the dominant over and understory plants of each forest ecosystem. SMBW forests dominated by AM plants had the highest levels of protein. BOWO forests with low AM plant cover had the lowest protein levels and SMRO forests were intermediate. Both Bradford-reactive fractions and their ratio varied seasonally, generally being highest in fall samples. Significant correlations observed between BRSP fractions, phosphorous, and soil organic matter were likely related to covariation of soil properties across forest types. While not statistically significant, response patterns of BRSP to N deposition were ecosystem-specific and reflected mycorrhizal types of dominant species. Abundance of these proteins reflected previously observed changes in SOC in the two forest types examined with abundant AM hosts. Specifically, nitrate addition led to BRSP decreases in SMBW and increases in SMRO forests. Changes in BRSP accounted for a small fraction of the changes in SOC; appearing to increase as a fraction of residual SOC consistent with the idea that GRSP are recalcitrant. BRSP remained unchanged at BOWO sites despite a significant increase in SOC at these sites. Our results point to the potential of proteins as contributors to differential, mycorrhizal type-specific responses to changes in soil carbon following N amendment.     

Spatial changes of arbuscular mycorrhizal fungi in peach and their correlation with soil properties

Arbuscular mycorrhizal (AM) fungi have beneficial effects on host plants, but their growth is influenced by various factors. This study was carried out to analyze the variation of AM fungi in soils and roots of peach (Prunus persica L. var. Golden Honey 3, a yellow-flesh variety) trees in different soil layers (0–40 cm) and their correlation with soil properties. The peach tree could be colonized by indigenous AM fungi (2.2–8.7 spores/g soil and 1.63–3.57 cm hyphal length/g soil), achieving 79.50–93.55% of root AM fungal colonization degree. The mycorrhizal growth, root sugars, soil three glomalins, NH₄⁺-N, NO₃⁻-N, available P and K, and soil organic matter (SOM) had spatial heterogeneity. Soil spores, but not soil hyphae contributed to soil glomalin, and soil glomalin also contributed to SOM. There was a significant correlation of soil hyphae with spore density, soil NO₃⁻-N, and SOM. Root mycorrhiza was positively correlated with spore density, NH₄⁺-N, NO₃⁻-N, and easily extractable glomalin-related soil protein. Notably, spore density positively correlated with NO₃⁻-N, available K, SOM, and root fructose and glucose, while negatively correlated with available P and root sucrose. These findings concluded that mycorrhiza of peach showed spatial distribution, and soil properties mainly affected/altered based on the soil spore density.     

荒漠油蒿根围AM真菌与球囊霉素的时空分布

2007年4月、7月和10月分别于陕西省榆林市北部沙地的油蒿(Artemisia.ordosica)根围分0～10 cm,10～20 cm,20～30 cm,30～40 cm和40～50 cm 5个土层采集土壤样品,系统研究了油蒿根围丛枝菌根(Arbuscular mycorrhiza,简称AM)真菌和球囊霉素的时空分布及与土壤因子的相关性.结果表明,油蒿根围AM真菌总定殖率为89.54%、泡囊定殖率为26.24%,丛枝定殖率为21.08%,孢子密度为2.91～6.17个/g土,说明油蒿能与AM真菌形成良好共生关系.从土壤样品中共分离出4属21种AM真菌,其中球囊霉属(Glomus)为优势属,地球囊霉 (G.geosporum)为优势种.油蒿根围AM真菌和球囊霉素具有明显的时空异质性,并与土壤因子密切相关.菌丝定殖率随季节变换逐渐增加,泡囊定殖率和丛枝定殖率在夏季最低,春秋相对较高,与孢子密度季相变化相反.油蒿根围总球囊霉素在0～20 cm 土层含量最高,随土层深度增加而递减.易提取球囊霉素含量随土层深度增加波动较大.球囊霉素春季含量最高,夏秋含量降低.总球囊霉素和易提取球囊霉素与土壤养分、土壤酶活性、AM真菌孢子密度均有极显著相关性,二者能综合反应土壤AM真菌群落、有机C动态和养分循环进程,应作为土壤质量及功能评价的新指标进一步深入研究.     

当归不同生长时期根际丛枝真菌分布及土壤养分和酶活性的动态变化

【目的】探讨当归不同生长时期丛枝菌根真菌(Arbuscular mycorrhizal fungi,AMF)的分布及土壤养分和土壤酶活的变化,以期了解当归不同生长时期AMF与土壤养分和土壤酶活的关系,为AMF在当归种植的应用提供理论依据。【方法】在当归不同生长时期分别采集根际土壤样品,测定其土壤养分、土壤酶活、AMF孢子密度和球囊霉素等因子,分析当归不同生长时期根际土壤AMF孢子密度、土壤养分和土壤酶活等指标的动态变化和相关性。【结果】随着当归生育期的完成,根际土壤AMF孢子密度先降低后持续升高;易提取球囊霉素(Easily extractable glomalin,EEG)和总球囊霉素(Total glomalin,TG)平稳增加,而且EET与TG和脲酶活性呈显著正相关(P0.01),EET和TG与根际土壤有机质和全氮含量以及酸性和中性磷酸酶活性均显著正相关(P0.05);根际土壤有机质和全氮表现为增加的总趋势;有效磷含量呈现为生长前期保持不变、中期显著降低、后期逐渐升高的趋势,而有效钾含量先逐渐增加,生长中后期显著降低的趋势;根际土壤酸性和中性磷酸酶活性均呈现逐渐增加趋势,而脲酶活性表现为生长前期逐渐增加,中后期显著降低;p H值在当归不同生长时期有所波动。相关性分析结果表明,AMF孢子密度与土壤酸性磷酸酶酶活性呈显著正相关,而酸性磷酸酶酶活性与根际土壤全氮、有机质、易提取球囊霉素和总球囊霉素呈显著正相关,与有效磷和有效钾含量呈显著负相关,表明AMF对根际土壤养分和酶活性具有一定的调节作用;主成分分析结果表明,不同生长时期是影响当归根际土壤理化指标的主要因素。【结论】AMF孢子密度在当归根际的动态变化一定程度上反映出了AMF分泌球囊霉素的能力,以及球囊霉素对增加根际土壤碳氮储存的贡献,同时表明球囊霉素影响了当归根际土壤酶活性和其它养分的代谢循环,对改良土壤和促进当归生长发挥重要的作用。     

A novel in vitro cultivation system to produce and isolate soluble factors released from hyphae of arbuscular mycorrhizal fungi

The Petri plate dual in vitro culture system for arbuscular mycorrhizal fungi (AMF) is ill-suited for the production and isolation of extraradically released soluble factors, thereby preventing the further characterization of such compounds. To overcome this technical bottleneck, we here describe a novel cultivation system using the standard AMF Glomus intraradices-carrot dual culture as a model. This new system is capable of producing soluble factors in copious amounts without any interference from host roots. The greatest advantages of this culture method include ease of handling and reusability of the culture vessel, thus making it a very cost effective system.