Polyploidy in tomato roots as affected by arbuscular mycorrhizal colonization

Nuclear changes in roots of tomato (Lycopersicon esculentum), a plant with a small genome, during the establishment of arbuscular mycorrhizal (AM) colonization were studied using light and electron microscopy, as well as flow and static cytometry. Nuclei of mycorrhizal root cortex cells were larger and had more decondensed chromatin than those of controls. Significant ploidy distribution differences were observed between nuclei of AM colonized and control roots, and a strong correlation between nuclear polyploidization and AM colonization was found. Polyploidization and decondensation are usually associated with high metabolic activity. The metabolic activity of mycorrhizal root cells, evaluated in this work as respiratory activity by using a cytochemical assay for succinate dehydrogenase combined with image analysis, increased in comparison to controls. The meaning of polyploidization is discussed in relation to the structural and metabolic modifications induced by mycorrhization.     

Apical meristems of tomato roots and their modifications induced by arbuscular mycorrhizal and soilborne pathogenic fungi

Functional morphological patterns in root apices of tomato ( Lycopersicon esculentum ) dependent on growth, ageing and infection by the arbuscular mycorrhizal (AM) fungus Glomus mosseae and/or by the soilborne pathogenic fungus Phytophthora nicotianae var parasitica ( P. parasitica ) were studied. Uninfected root apices were characterized by closed, tri-layered meristems with nonreticulate nuclei; however, some apices of each treatment lost their meristematic nature, stopped growing and differentiated, becoming 'parenchymatized'. The pathogenic fungus reduced the apex diameter and the number of mitotically active and viable apices inducing plasmolysis, cell and nucleus degeneration, and necrosis. The AM fungus, on the other hand, produced an increase in apex size and reduced the percentage of necrosis both in uninfected roots and in roots infected by P. parasitica . Thus, the AM fungus protected the apices from the pathogenic infection, allowing normal root growth. Furthermore, larger apices, which produce thicker roots, might indirectly contribute to plant protection. Increased volumes of colonizable tissues favour the spreading of the symbiont, and P. parasitica hyphae are always excluded from arbuscule-containing cells.     

Comparison of morphological and molecular genetic quantification of relative abundance of arbuscular mycorrhizal fungi within roots

Nested PCR amplicons of ribosomal RNA genes have been used to identify individuals within assemblages of arbuscular mycorrhizal (AM) fungi in roots and to estimate their relative abundance. Microscopy has also been used to identify their relative abundance in roots, but only at low resolution, usually the genus level. We evaluated the robustness of using nested PCR amplicons of ribosomal RNA genes to estimate the relative abundance of undefined AM fungi in uniformly aged roots in comparison to visual estimates. The relative abundance of AM fungi was assessed as per cent root length colonised by morphotypes and relative sequence type abundance in clone libraries. Plants were grown in coastal soil to obtain assemblages of unknown AM fungi at two times (spring and autumn). Relative abundance of dominant genera of AM fungi in roots (Archaeospora and Glomus) based on an analysis of ribosomal RNA genes did not consistently correspond with relative abundance of morphotypes. This microscopic vs. molecular genetic comparison supports previous conclusions that there can be limitations in using nested PCR amplicons for quantifying the relative abundance of AM fungi in roots, with a sampling bias likely to be of significance. Both molecular genetic and morphological methods are used to estimate relative abundance of AM fungi as a precursor to understanding mycorrhizal function in field soils, but they are rarely verified using alternative approaches although this may be necessary.     

Chitosanase and chitinase activities in tomato roots during interactions with arbuscular mycorrhizal fungi or Phytophthora parasitica

New chitosanase acidic isoforms have been shown in Glomus mosseae-colonized tomato roots and their induction, together with the previously described mycorrhiza-related chitinase isoform, has been further corroborated in plants colonized with another Glomus species (G. intraradices),as well as in tomato roots colonized in vitro by Giaspora rosea. The induction of these chitosanase isoforms appears as a specific response to the arbuscular mycorrhizal (AM) symbiosis, and does not correspond to unspecific defence mechanisms, since these isoforms were not induced by the pathogen Phytophthora parasitica. Analysis by isoelectrofocusing showed two closely migrating chitinase isoforms, specific to mycorrhizal plants colonized either with G. mosseae or G. intraradices, and their isoelectric points were estimated to be 4.5 and 4.7. The estimated molecular mass of chitosanases was 20 kDa, and after isoelectrofocusing, the chitosanase activities were detected along the acidic pH range (6.5-3.5). Constitutive and induced isoforms were also investigated during a time-course study. In some experiments, chitin and chitosan were embedded together as substrates in polyacrylamide gels with the aim of studying the capacity of some isoforms to display both chitinase and chitosanase activities. In extracts from plants colonized with either G. mosseae or G. intraradices, some constitutive chitinases and the previously described mycorrhiza-related chitinase isoform, appeared to display chitosanase activity, while this bifunctional character was not found for the chitinases from non-mycorrhizal tissue, nor in Phytophthora-infected plants. These results suggest some diversity in the chitinase activities concerning substrate specificity in mycorrhizal plants. The possible implications of these observations in the functioning of the symbiosis is discussed.Key words: Arbuscular mycorrhizas, chitinases, chitosanases, Phytophthora parasitica, tomato, Lycoperiscon esculentum.      

Linking lipid transfer with reduced arbuscule formation in tomato roots colonized by arbuscular mycorrhizal fungus under low pH stress

Arbuscules are the core structures of arbuscular mycorrhizae (AM), and arbuscule development is regulated by environmental stress, e.g., low pH. Recent studies indicate that lipid transfer from plants is essential for AM fungal colonization; however, the role of lipid transfer in arbuscule formation and the dynamics of lipid accumulation in arbuscules under low pH stress are far from well understood. In the symbiosis of tomato and Rhizophagus intraradices under contrasting pH conditions (pH 4.5 vs. pH 6.5), we investigated arbuscule formation, nutrient uptake, alkaline phosphatase activity and lipid accumulation; examined the gene expression involved in phosphate transport, lipid biosynthesis and transfer and sugar metabolism; and visualized the lipid dynamics in arbuscules. Low pH greatly inhibited arbuscule formation, in parallel with reduced phospholipid fatty acids accumulation in AM fungus and decreased P uptake. This reduction was supported by the decreased expression of plant genes encoding lipid biosynthesis and transfer. More degenerating arbuscules were observed under low pH conditions, and neutral lipid fatty acids accumulated only in degenerating arbuscules. These data reveal that, under low pH stress, reduced lipid transfer from hosts to AM fungi is responsible for the inhibited arbuscule formation.     

Polyphosphate dynamics in mycorrhizal roots during colonization of an arbuscular mycorrhizal fungus

Inorganic polyphosphate (poly P) has been considered to be a translocatable form of phosphate (Pi) in arbuscular mycorrhizal fungi (AMF). Here we examined time-course changes in poly P content during the AMF colonization process. Onion (Allium cepa) plants were cultured with or without inoculation with Gigaspora margarita for 2-8 wk with periodic sampling. Poly P in the extracts, purified through gel filtration, was quantified by the reverse reaction of polyphosphate kinase. The length of poly P in mycorrhizal roots appeared to be shorter than in extraradical hyphae or in spores of the AMF, indicating that AMF depolymerize poly P before providing Pi to the host. The poly P content increased as colonization proceeded, and was highly correlated with the weight of the colonized roots. These results support the model that AMF supply Pi to the host through the poly P pool, and that the poly P content of a mycorrhizal root can be a good indicator of the Pi-supplying activity of AMF.     

Gibberellin metabolism: new insights revealed by the genes

The identification of most of the genes involved in the metabolic pathways for gibberellin hormones has helped us to understand these pathways and their regulation. Many of these enzymes are multifunctional and therefore fewer enzymes than might be expected are required to synthesize the various gibberellin structures. However, several of the enzymes are encoded by multiple genes that are regulated differently, adding unexpected genetic complexity. Several endogenous and environmental factors modify the expression of gibberellin biosynthesis genes, including developmental stage, hormonal status and light. A future challenge will be to dissect the complex, interacting pathways that mediate the regulation of gibberellin metabolism.     

丛枝菌根真菌侵染番茄离体毛状根双重培养体系的建立

利用发根农杆菌Agrobacterium rhizogenes A4诱导樱桃番茄Micro-Tom产生毛状根,并在此基础上成功建立了丛枝菌根(AM)真菌根内球囊霉Glomus intraradices与转移Ri T-DNA番茄离体毛状根的双重培养体系。该真菌侵染14d左右,菌丝开始形成多级分支,17-21d时,一些菌丝顶端膨大,长出新生孢子。接种后3个月时,每皿孢子数量达到600-800个。新形成的孢子无需休眠,可直接侵染番茄离体根。成功的番茄双重培养为番茄菌根生理分子机制的研究提供了理想的实验体系。     

New insights into the signaling pathways controlling defense gene expression in rice roots during the arbuscular mycorrhizal symbiosis

Mycorrhizal fungi form a mutualistic relationship with the roots of most plant species. This association provides the arbuscular mycorrhizal (AM) fungus with sugars while the fungus improves the uptake of water and mineral nutrients in the host plant. Moreover, the induction of defense gene expression in mycorrhizal roots has been described. While salicylic acid (SA)-regulated Pathogenesis-Related (PR) proteins accumulate in rice roots colonized by the AM fungus G. intraradices , the SA content is not significantly altered in the mycorrhizal roots. Sugars, in addition to being a source of carbon for the fungus, might act as signals for the control of defense gene expression. We hypothesize that increased demands for sugars by the fungus might be responsible for the activation of the host defense responses which will then contribute to the stabilization of root colonization by the AM fungus. An excessive root colonization might change a mutualistic association into a parasitic association.Key words: Glomus intraradices, glucose, fructose, Oryza sativa, pathogenesis-related (PR), salicylic acid (SA), sucrose, sugarsThe arbuscular mycorrhizal (AM) fungi are obligate biotrophs that establish mutualistic associations with the roots of over 90% of all plant species. AM fungi improve the uptake of water and mineral nutrients in the host plant, mainly phosphorus and nitrogen, in exchange for sugars generated from photosynthesis. The benefits of the AM symbiosis on plant fitness are largely known, including increased ability to cope with biotic and abiotic stresses.^1,2 In fact, the amount of carbon allocated to mycorrhizal roots might be up 20% of the total photosynthate income.³ During root colonization, the AM fungus penetrates into the root through the epidermal cells and colonizes the cortex. In the root cortical cells, the fungus forms highly branched structures, called arbuscules, which are the site of the major nutrient exchange between the two symbionts.^4,5 The legumes Medicago truncatula and Lotus japonicus have been widely adopted as the reference species for studies of the AM symbiosis. Cereal crops and rice in particular are also able to establish symbiotic associations with AM fungi.^6,7 Arabidopsis thaliana, the model system for functional genomics in plants, has no mycorrhization ability.It is also well known that plants have evolved inducible defense systems to protect themselves from pathogen invasion. Challenge with a pathogen activates a complex variety of defense reactions that includes the rapid generation of reactive oxygen species (ROS), changes in ion fluxes across the plasma membrane, cell wall reinforcement and production of antimicrobial compounds (e.g., phytoalexins).⁸ One of the most frequently observed biochemical events following pathogen infection is the accumulation of pathogenesis-related (PR) proteins.⁹ For some PR proteins antimicrobial activities have been described (e.g., chitinases, β-1,3-glucanases, thionins or defensins). The plant responses to pathogen attack are activated both locally and systemically. The phytohormones salicyclic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) act as defense signaling molecules for the activation of defense responses.¹⁰ Whereas SA-dependent signaling often provides resistance to biotrophic pathogens, JA/ET-dependent signaling is effective against necrotrophic pathogens.¹¹ During plant-pathogen interactions, cross-talk between SA and JA/ET signaling pathways provides the plant with the opportunity to prioritize one pathway over another to efficiently fine-tune its defense response to the invading pathogen. Contrary to biotrophic pathogens which exhibit a high degree of host specificity, the AM fungi manage to colonize a broad range of plant species.Evidence also exists on the existence of common mechanisms and signaling pathways governing responses to AM and pathogenic fungi.^2,12,13 Alterations in the content of hormones acting as defense signals also appear to occur during the AM symbiosis. As an example, JA and its derivatives (jasmonates) are believed to play an important role during the AM symbiosis in M. truncatula or tomato plants.^14,15 However, controversial data exists in the literature concerning the involvement of the various defense-related hormones during AM functioning. In particular, our current understanding of SA signaling during AM symbiosis is not clear.We recently documented the symbiotic proteome of the rice roots during their interaction with the AM fungus Glomus intraradices.⁶ A majority of the proteins identified in the rice symbiotic proteome are proteins with a function in defense responses or sugar metabolism. Among the proteins that accumulated at high levels in mycorrhizal rice roots compared to non mycorrhizal roots were PR proteins belonging to different PR families, such as PR1, chitinases (PR3), PR5 and several PR10 proteins. The PR1 and PBZ1 (a member of the PR10 family of PR proteins) genes are considered markers of the activation of defense responses in rice plants.^16,17 Of interest, the expression of many of the AM-regulated PR genes was previously reported to be induced by SA.^16,18–20 Proteins acting as oxidative stress protectors, such as ascorbate peroxidases, peroxidases and glutathione-S-transferases, also accumulated in mycorrhizal rice roots. Together, these observations support that the plant''s immune system is activated in the mycorrhizal rice root.To gain further insights into the molecular mechanisms governing PR gene expression in mycorrhizal roots, the SA and sugar contents of mycorrhizal roots were determined. Towards this end, rice (Oryza sativa ssp. japonica cv. Senia) plants were inoculated with the AM fungus G. intraradices.⁶ At 42 days post-inoculation (dpi), the overall colonization of the rice roots ranged from 25 to 30% as judged by microscopical observations of trypan blue-stained roots (results not shown; similar results were reported previously in reference ⁶). By this time, all the events related to fungal development, namely intraradical hyphae, arbuscules at different morphological stages of formation and vesicles, were present in G. intraradices-inoculated roots, thus confirming the establishment of the symbiotic association in the rice roots.Knowing that many AM-regulated proteins are also regulated by SA in rice roots, it was of interest to determine whether the level of endogenous SA increases in mycorrhizal roots compared to non mycorrhizal roots. In plants, intracellular SA is found predominantly as free SA and its sugar conjugate SA-glucoside (SAG). Root samples were analyzed for SA content, by measuring the level of both free SA and SAG as previously described in reference ²¹. This analysis revealed no significant differences, neither in free nor in SAG, between mycorrhizal and non mycorrhizal roots (Fig. 1). Then, it appears that although the expression of PR genes (functioning in a SA-dependent manner) is activated during the AM symbiosis, the fungus G. intraradices do not exploit the SA-mediated signaling pathway for induction of PR genes.Open in a separate windowFigure 1SA content, free SA and SA-glucoside (SAG) conjugate, in roots of mock-inoculated (−Gi) and G. intraradices-inoculated (+Gi) rice plants. SA determination was carried out at 42 days post-inoculation with G. intraradices. Three independent biological samples and three replicates per biological sample were used for quantification of SA. Two out of the three samples were the same ones used for the characterization of the symbiotic proteome in which the accumulation of SA-regulated PR genes was observed in reference ⁶. FW, fresh weight. Bars represent the means ± standard error.On the other hand, a direct link between sugar metabolism and the plant defense response has been established, including the phenomenon of high sugarmediated resistance and the finding that various key PR genes are induced by sugars. Transgenic approaches that lead to alterations in photoassimilate partitioning, either sucrose or hexoses, also alter PR gene expression.^22,23 In other studies, a SA-independent induction of PR genes by soluble sugars, sucrose, glucose and fructose, was reported in reference ²⁴.Sucrose, the main form of assimilated carbon during photosynthesis, is transported to the root tissues via the phloem where it becomes available to the root cells. As previously mentioned, characterization of the rice symbiotic proteome revealed alterations in the accumulation of proteins involved in sugar metabolism, such as enzymes involved in glucolysis/gluconeogenesis (e.g., fructose-1,6-bisphophate aldolase, enolase) or in pentose interconversions (e.g., UDP-glucose dehydrogenase).⁶ Because the plant provides sugars to the fungus, it is not surprising to find alterations in enzymes involved in sugar metabolism in the mycorrhizal roots. Evidence also supports that AM fungi acquire hexoses from the host cell and transform it into trehalose and glycogen, the typical sugars in the fungus.²⁵ Utilization of sucrose then requires hydrolysis in the plant cell which can be performed by sucrose synthase, producing UDP-glucose and fructose or invertases, producing glucose and fructose. Along with this, increased activities of invertases and sucrose synthases or increased expression of their corresponding genes, have been described during AM symbiotic interactions.^26,27 Very recently, the MtSucS1 sucrose synthase gene was reported to be essential for the establishment and maintenance of the AM symbiosis in Medicago truncatula.²⁸ In this context, we decided to explore whether colonization by G. intraradices has an effect on the accumulation of soluble sugars in rice roots.Sucrose, glucose and fructose content were measured enzymatically²³ in the rice roots at 42 days post-inoculation with G. intraradices . A tendency to a higher sucrose level was observed in mycorrhizal roots compared to non-mycorrhizal roots (Fig. 2). Concerning the hexose content, the mycorrhizal roots had a significantly lower hexose, both glucose and fructose levels, compared to non-mycorrhizal roots (p ≤ 0.05, Fig. 2). This finding is in agreement with results reported by other authors indicating that the fungal symbiont takes up and uses hexoses within the root.^29,30 The observation that the sucrose content is not significantly affected by mycorrhiza functioning, indicates that the host cell is able to sense sucrose concentration in order to maintain it at sufficient but constant levels to satisfy the demand for sugars by the fungal symbiont.Open in a separate windowFigure 2Sugar content in roots of rice plants inoculated with G. intraradices (+Gi) or mock-inoculated (−Gi). (A) Sucrose content. (B) Glucose content. (C) Fructose content. Measurements were made at 42 days post-inoculation with G. intraradices. Bars represent the means ± standard error.Clearly, the outcome of the AM symbiosis is an overall improvement of the fitness of both partners: the plant supplies the fungus with photosynthates whereas the fungus delivers nutrients from the soil to the host plant. Variations in the extent of colonization of the AM fungi will impose different carbon demands on the plants. However, a high demand of photosynthates by the mycorrhizal root might result in increased mycorrhization which, in turn, might be detrimental for the host plant. The rate of colonization and the amount of fungal biomass must then be tightly controlled by the host plant. We postulate that an increased sink strength by AM colonization might result in transient and/or localized increases in sugar concentrations in the root cell which might be the signal for the activation of defense gene expression. A schematic representation of plant responses associated with increased demands for sugars and deployment of defense responses is shown in Figure 3. According to this model, sugars might play a dual role during the AM symbiosis: (1) sugars are transferred from the plant to the fungus in exchange of mineral nutrients and (2) sugars alter host gene expression, leading to the activation of defense-related genes. This will allow the host plant to avoid an excessive root colonization by the AM fungus that might cause negative effects on the plant''s fitness. A complex exchange and interplay of signals between plant roots and AM fungi must then operate during functioning of the AM symbiosis for coordination of joint nutrient resource explotation strategies and control of the plant''s immune system. During evolution, co-adaptation between the two symbionts, the AM fungi and the host plant, must have occurred for stabilization of mycorrhizal cooperation and optimal functioning of mycorrhizal associations along the mutualism-parasitism continuum.Open in a separate windowFigure 3Proposed model for a sugar mediated-activation of defense-related genes in mycorrhizal roots. In the arbuscular mycorrhizal symbiosis, the fungal symbiont colonizes root cortical cells, where it establishes differentiated hyphae called arbuscules. Arbuscules are the site of mineral nutrient transfer to the plant and the site of carbon acquisition by the fungus. Although arbuscules form within the root cortical cells, they remain separated from the plant cell cytoplasm by a plant-derived membrane, the periarbuscular membrane. In this way, an interface is created between the plant and fungal cells which appears to be optimal for nutrient transfer. Sucrose is transported through the phloem into the root. In the root cell, sucrose is hydrolyzed by host invertase and sucrose synthase activities before uptake by the AM fungus. Hexose uptake at the plant-fungus interfase might be passive with a concentration gradient maintained by rapid conversion of hexoses taken up by the fungus to trehalose and glycogen. Active mechanisms might also operate for hexose transport processes between the host cell and the symbiont. Under conditions of a high demand for sugars by the AM fungus, transient increases in sugar content will occur in the root cells which would be the signal for the activation of the host defense responses. The host-produced defense compounds would stabilize the level of root colonization by the AM fungus. An excessive root colonization might change the mutualistic association into a parasitic one.     

Elemental composition in vesicles of an arbuscular mycorrhizal fungus, as revealed by PIXE analysis

We investigated element accumulation in vesicles of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, extracted from the roots of inoculated leek plants. The elemental composition (elements heavier than Mg) was quantified using particle-induced X-ray emission (PIXE), in combination with scanning transmission ion microscopy (STIM). In vesicles, P was the most abundant of the elements analysed, followed by Ca, S, Si and K. We analysed 12 vesicles from two root systems and found that the variation between vesicles was particularly high for P and Si. The P content related positively to Si, Zn and K, while its relation to Cl fitted to a negative power function. Vesicle transects showed that P and K were present in central parts, while Ca was present mainly near the vesicle surfaces. The results showed that P is an important part (0.5% of the dry weight) of the vesicle content and that the distribution of some elements, within mycelia, may be strongly correlated.     

Kinetics of phosphorus absorption by mycorrhizal and nonmycorrhizal tomato roots

Kinetics of P absorption were investigated in mycorrhizal (Glomus fasciculatus) and nonmycorrhizal tomato (Lycopersicon esculentum) roots to determine why increased ion absorption by mycorrhizae occurs. Initial rates of absorption of ³²P were measured at 1 to 100 micromolar KH₂PO₄ (pH 4.6). Absorption rates of mycorrhizae were about twice those of control roots. Augustinsson-Hofstee analysis yielded two linear phases; V_max and K_m were calculated for each phase. In the low phase (1 to 20 micromolar), V_max values for the mycorrhizal and nonmycorrhizal roots were each 0.10 micromoles P per gram fresh weight per hour while K_m values were 1.6 and 3.9 micromolar KH₂PO₄, respectively. For the high phase (30 to 100 micromolar), V_max values for mycorrhizal and nonmycorrhizal roots were 0.32 and 0.25 micromoles P per gram fresh weight per hour and K_m values were 35 and 42 micromolar, respectively. These results indicate that at the lower phase concentrations, similar to those expected in most soil solutions, a major factor contributing to the increased uptake was an apparent greater affinity of the absorbing sites for H₂PO₄⁻ (lower K_m).