Impact of sewage sludges on Medicago truncatula symbiotic proteome

The effects of sewage sludges were investigated on the symbiotic interactions between the model plant Medicago truncatula and the arbuscular mycorrhizal fungus Glomus mosseae or the rhizobial bacteria Sinorhizobium meliloti. By comparison to a control sludge showing positive effects on plant growth and root symbioses, sludges enriched with polycylic aromatic hydrocarbons or heavy metals were deleterious. Symbiosis-related proteins were detected and identified by two-dimensional electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, and image analysis was used to study the effects of sewage sludges on M. truncatula symbiotic proteome.     

Sub-cellular proteomic analysis of a Medicago truncatula root microsomal fraction

Since the last decade, Medicago truncatula has emerged as one of the model plants particularly investigated in the field of plant-microbe interactions. Several genetic and molecular approaches including proteomics have been developed to increase knowledge about this plant species. To complement the proteomic data, which have mainly focused on the total root proteins from M. truncatula, we carried out a sub-cellular approach to gain access to the total membrane-associated proteins. Following the setting up of the purification process, microsomal proteins were separated on 2-DE. Ninety-six out of the 440 well-resolved proteins were identified by MALDI-TOF peptide mass fingerprinting. A high percent (83%) of successful protein identification was obtained when using M. truncatula clustered EST database for queries. During the purification process, the enrichment in membrane-associated proteins was monitored on 2-D gels. The membrane location of microsomal proteins was further confirmed using PMF identification. This study reports a fractionation process for characterizing microsomal root proteins of M. truncatula, which could be an interesting tool for investigating the molecular mechanisms involved in root symbioses.     

摩西球囊霉对三叶鬼针草保护酶活性的影响

旱地农田入侵杂草三叶鬼针草(Bidens pilosa L.)与摩西球囊霉(Glomus mosseae)(AM真菌)经常形成长效的共生体,该霉菌对三叶鬼针草的入侵能力起到促进作用,但机理并不清楚。盆栽试验对正常浇水、中度干旱和重度干旱条件下接种AM真菌的三叶鬼针草植株与未接种植株之间叶片丙二醛(MDA)含量及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸氧化酶(ASP)和过氧化物酶(POD)等保护酶活性进行了比较研究。结果表明,干旱胁迫导致三叶鬼针草叶片内MDA含量升高,SOD、CAT、ASP和POD的活性升高;正常浇水条件下,接种G. mosseae 对MDA含量,SOD、ASP和CAT活性影响不显著;中度干旱条件下,接种没有显著影响ASP活性,但对SOD和CAT活性影响显著;在处理前期(7,14,21d)POD活性影响不显著,在处理后期(28,35d)接种植株显著低于未接种植株;重度干旱条件下,未接种植株MDA含量、CAT活性显著高于接种植株,POD活性差异不显著。ASP活性在21d前差异不显著,之后,未接种植株显著高于接种植株。因此,AM真菌G. mosseae 有效地降低了干旱胁迫对三叶鬼针草的伤害程度,随着土壤含水量的严重亏缺和胁迫时间的延长,摩西球囊霉对三叶鬼针草的保护作用逐渐减弱。由于三叶鬼针草和AM真菌之间普遍存在着共生关系,该共生关系可能是三叶鬼针草入侵能力强的关键生物因子之一。     

Aquaporin genes GintAQPF1 and GintAQPF2 from Glomus intraradices contribute to plant drought tolerance

Arbuscular mycorrhizal (AM) symbiosis, established between AM fungi (AMF) and roots of higher plants, occurs in most terrestrial ecosystems. It has been well demonstrated that AM symbiosis can improve plant performance under various environmental stresses, including drought stress. However, the molecular basis for the direct involvement of AMF in plant drought tolerance has not yet been established. Most recently, we cloned two functional aquaporin genes, GintAQPF1 and GintAQPF2, from AM fungus Glomus intraradices. By heterologous gene expression in yeast, aquaporin localization, activities and water permeability were examined. Gene expressions during symbiosis in expose to drought stress were also analyzed. Our data strongly supported potential water transport via AMF to host plants. As a complement, here we adopted the monoxenic culture system for AMF, in which carrot roots transformed by Ri-T DNA were cultured with Glomus intraradices in two-compartment Petri dishes, to verify the aquaporin gene functions in assisting AMF survival under polyethylene glycol (PEG) treatment. Our results showed that 25% PEG significantly upregulated the expression of two aquaporin genes, which was in line with the gene functions examined in yeast. We therefore concluded that the aquaporins function similarly in AMF as in yeast subjected to osmotic stress. The study provided further evidence to the direct involvement of AMF in improving plant water relations under drought stresses.     

Abscisic acid rescues the root meristem defects of the Medicago truncatula latd mutant

The LATD gene of the model legume, Medicago truncatula, is required for the normal function of three meristems, i.e. the primary root, lateral roots and nitrogen-fixing nodules. In latd mutants, primary root growth eventually arrests, resulting in a disorganized root tip lacking a presumptive meristem and root cap columella cells. Lateral root organs are more severely affected; latd lateral roots and nodules arrest immediately after emerging from the primary root, and reveal a lack of organization. Here we show that the plant hormone, abscisic acid (ABA), can rescue the latd root, but not nodule, meristem defects. Growth on ABA is sufficient to restore formation of small, cytoplasm-rich cells in the presumptive meristem region, rescue meristem organization and root growth and formation of root cap columella cells. In contrast, inhibition of ethylene synthesis or signaling fails to restore latd primary root growth. We find that latd mutants have normal levels of ABA, but exhibit reduced sensitivity to the hormone in two other ABA-dependent processes: seed germination and stomatal closure. Together, these observations demonstrate that the latd mutant is defective in the ABA response and indicate a role for LATD-dependent ABA signaling in M. truncatula root meristem function.     

Absence of carbon transfer between Medicago truncatula plants linked by a mycorrhizal network, demonstrated in an experimental microcosm

Carbon transfer between plants via a common extraradical network of arbuscular mycorrhizal (AM) fungal hyphae has been investigated abundantly, but the results remain equivocal. We studied the transfer of carbon through this fungal network, from a Medicago truncatula donor plant to a receiver (1) M. truncatula plant growing under decreased light conditions and (2) M. truncatula seedling. Autotrophic plants were grown in bicompartmented Petri plates, with their root systems physically separated, but linked by the extraradical network of Glomus intraradices. A control Myc-/Nod- M. truncatula plant was inserted in the same compartment as the receiver plant. Following labeling of the donor plant with 13CO2, 13C was recovered in the donor plant shoots and roots, in the extraradical mycelium and in the receiver plant roots. Fatty acid analysis of the receiver's roots further demonstrated 13C enrichment in the fungal-specific lipids, while almost no label was detected in the plant-specific compounds. We conclude that carbon was transferred from the donor to the receiver plant via the AM fungal network, but that the transferred carbon remained within the intraradical AM fungal structures of the receiver's root and was not transferred to the receiver's plant tissues.     

OsIPD3, an ortholog of the Medicago truncatula DMI3 interacting protein IPD3, is required for mycorrhizal symbiosis in rice

Medicago truncatula IPD3 (MtIPD3) is an interacting protein of DMI3 (does not make infections 3), a Ca(2+)/calmodulin-dependent protein kinase (CCaMK) essential for both arbuscular mycorrhizal (AM) and rhizobial symbioses. However, the function of MtIPD3 in root symbioses has not been demonstrated in M. truncatula, because of a lack of knockout mutants for functional analysis. In this study, the availability of IPD3 knockout mutants in rice (Oryza sativa) was exploited to test the function of OsIPD3 in AM symbiosis. Three independent retrotransposon Tos17 insertion lines of OsIPD3 were selected and the phenotypes characterized upon inoculation with the AM fungus Glomus intraradices. Phenotypic and genetic analyses revealed that the Osipd3 mutants were unable to establish a symbiotic association with G. intraradices. In conclusion, IPD3 represents a novel gene required for root symbiosis with AM fungi in plants.     

Suppression of allene oxide cyclase in hairy roots of Medicago truncatula reduces jasmonate levels and the degree of mycorrhization with Glomus intraradices

During the symbiotic interaction between Medicago truncatula and the arbuscular mycorrhizal (AM) fungus Glomus intraradices, an endogenous increase in jasmonic acid (JA) occurs. Two full-length cDNAs coding for the JA-biosynthetic enzyme allene oxide cyclase (AOC) from M. truncatula, designated as MtAOC1 and MtAOC2, were cloned and characterized. The AOC protein was localized in plastids and found to occur constitutively in all vascular tissues of M. truncatula. In leaves and roots, MtAOCs are expressed upon JA application. Enhanced expression was also observed during mycorrhization with G. intraradices. A partial suppression of MtAOC expression was achieved in roots following transformation with Agrobacterium rhizogenes harboring the MtAOC1 cDNA in the antisense direction under control of the cauliflower mosaic virus 35S promoter. In comparison to samples transformed with 35SuidA, roots with suppressed MtAOC1 expression exhibited lower JA levels and a remarkable delay in the process of colonization with G. intraradices. Both the mycorrhization rate, quantified by fungal rRNA, and the arbuscule formation, analyzed by the expression level of the AM-specific gene MtPT4, were affected. Staining of fungal material in roots with suppressed MtAOC1 revealed a decreased number of arbuscules, but these did not exhibit an altered structure. Our results indicate a crucial role for JA in the establishment of AM symbiosis.     

Recent Progress in Development of Tnt1 Functional Genomics Platform for Medicago truncatula and Lotus japonicus in Bulgaria

Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project 'GLIP': Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project 'IFCOSMO': Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.     

Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism

Lotus japonicus and Medicago truncatula model legumes, which form determined and indeterminate nodules, respectively, provide a convenient system to study plant-Rhizobium interaction and to establish differences between the two types of nodules under salt stress conditions. We examined the effects of 25 and 50mM NaCl doses on growth and nitrogen fixation parameters, as well as carbohydrate content and carbon metabolism of M. truncatula and L. japonicus nodules. The leghemoglobin (Lb) content and nitrogen fixation rate (NFR) were approximately 10.0 and 2.0 times higher, respectively, in nodules of L. japonicus when compared with M. truncatula. Plant growth parameters and nitrogenase activity decreased with NaCl treatments in both legumes. Sucrose was the predominant sugar quantified in nodules of both legumes, showing a decrease in concentration in response to salt stress. The content of trehalose was low (less than 2.5% of total soluble sugars (TSS)) to act as an osmolyte in nodules, despite its concentration being increased under saline conditions. Nodule enzyme activities of trehalose-6-phosphate synthase (TPS) and trehalase (TRE) decreased with salinity. L. japonicus nodule carbon metabolism proved to be less sensitive to salinity than in M. truncatula, as enzymatic activities responsible for the carbon supply to the bacteroids to fuel nitrogen fixation, such as sucrose synthase (SS), alkaline invertase (AI), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC), were less affected by salt than the corresponding activities in barrel medics. However, nitrogenase activity was only inhibited by salinity in L. japonicus nodules.