Using the Maize Nested Association Mapping (NAM) Population to Partition Arbuscular Mycorrhizal Effects on Drought Stress Tolerance into Hormonal and Hydraulic Components.

In this study, a first experiment was conducted with the objective of determining how drought stress alters the radial water flow and physiology in the whole maize nested association mapping (NAM) population and to find out which contrasting maize lines should be tested in a second experiment for their responses to drought in combination with an arbuscular mycorrhizal (AM) fungus. Emphasis was placed on determining the role of plant aquaporins and phytohormones in the responses of these contrasting maize lines to cope with drought stress. Results showed that both plant aquaporins and hormones are altered by the AM symbiosis and are highly involved in the physiological responses of maize plants to drought stress. The regulation by the AM symbiosis of aquaporins involved in water transport across cell membranes alters radial water transport in host plants. Hormones such as IAA, SA, ABA and jasmonates must be involved in this process either by regulating the own plant-AM fungus interaction and the activity of aquaporins, or by inducing posttranscriptional changes in these aquaporins, which in turns alter their water transport capacity. An intricate relationship between root hydraulic conductivity, aquaporins and phytohormones has been observed, revealing a complex network controlling water transport in maize roots.

Na+ transporter HKT1;2 reduces flower Na+ content and considerably mitigates the decline in tomato fruit yields under saline conditions.

Genes encoding HKT1-like Na+ transporters play a key role in the salinity tolerance mechanism in Arabidopsis and other plant species by retrieving Na+ from the xylem of different organs and tissues. In this study, we investigated the role of two HKT1;2 allelic variants in tomato salt tolerance in relation to vegetative growth and fruit yield in plants subjected to salt treatment in a commercial greenhouse under real production conditions. We used two near-isogenic lines (NILs), homozygous for either the Solanum lycopersicum (NIL17) or S. cheesmaniae (NIL14) allele, at HKT1;2 loci and their respective RNAi-Sl/ScHKT1;2 lines. The results obtained show that both ScHKT1;2- and SlHKT1;2-silenced lines display hypersensitivity to salinity associated with an altered leaf Na+/K+ ratio, thus confirming that HKT1;2 plays an important role in Na+ homeostasis and salinity tolerance in tomato. Both silenced lines also showed Na+ over-accumulation and a slight, but significant, reduction in K+ content in the flower tissues of salt-treated plants and consequently a higher Na+/K+ ratio as compared to the respective unsilenced lines. This altered Na+/K+ ratio in flower tissues is associated with a sharp reduction in fruit yield, measured as total fresh weight and number of fruits, in both silenced lines under salinity conditions. Our findings demonstrate that Na+ transporter HKT1;2 protects the flower against Na+ toxicity and mitigates the reduction in tomato fruit yield under salinity conditions.

Arbuscular Mycorrhizal Fungal Gene Expression Analysis by Real-Time PCR.

Gene expression analysis is a broadly used and powerful technique in many fields of biological research. The expression pattern of specific marker genes provides an insight into complex regulatory networks and leads to the identification of relevant genes associated to specific biological processes, such as arbuscular mycorrhizal symbiosis. Among the existing gene expression analysis toolbox, reverse transcriptase coupled to quantitative polymerase chain reaction (qRT-PCR) is considered the gold standard for accurate, sensitive, fast, and relatively inexpensive measurement. However, for a correct identification of differentially expressed genes, appropriate controls are required in order to minimize nonspecific variations associated with intrinsic technical variability. In this chapter, we recommend a number of tips to use qRT-PCR analysis in mycorrhizal roots and fungal mycelium.

Expression analysis and functional characterization of two PHT1 family phosphate transporters in ryegrass.

MAIN CONCLUSION: The phosphate transporters LpPHT1;1 and LpPHT1;4 have different roles in phosphate uptake and translocation in ryegrass under P stress condition. The phosphate transporter 1 (PHT1) family are integral membrane proteins that operate in phosphate uptake, distribution and remobilization within plants. In this study, we report on the functional characterization and expression of two PHT1 family members from ryegrass plants (Lolium perenne L.) and determine their roles in the specificity of Pi transport. The expression level of LpPHT1;4 was strongly influenced by phosphorus (P) status, being higher under P-starvation condition. In contrast, the expression level of LpPHT1;1 was not correlated with P supply. Yeast mutant complementation assays showed that LpPHT1;4 can complement the growth defect of the yeast mutant Δpho84 under Pi-deficient conditions, whereas the yeast mutant expressing LpPHT1;1 was not able to restore growth. Phylogenetic and molecular analyses indicated high sequence similarity to previously identified PHT1s from other species in the Poaceae. These results suggest that LpPHT1;1 may function as a low-affinity Pi transporter, whereas LpPHT1;4 could acts as a high-affinity Pi transporter to maintain Pi homeostasis under stress conditions in ryegrass plants. This study will form the basis for the long-term goal of improving the phosphate use efficiency of ryegrass plants.

Review: Arbuscular mycorrhizas as key players in sustainable plant phosphorus acquisition: An overview on the mechanisms involved.

Phosphorus (P) is a poorly available macronutrient essential for plant growth and development and consequently for successful crop yield and ecosystem productivity. To cope with P limitations plants have evolved strategies for enhancing P uptake and/or improving P efficiency use. The universal 450-million-yr-old arbuscular mycorrhizal (AM) (fungus-root) symbioses are one of the most successful and widespread strategies to maximize access of plants to available P. AM fungi biotrophically colonize the root cortex of most plant species and develop an extraradical mycelium which overgrows the nutrient depletion zone of the soil surrounding plant roots. This hyphal network is specialized in the acquisition of low mobility nutrients from soil, particularly P. During the last years, molecular biology techniques coupled to novel physiological approaches have provided fascinating contributions to our understanding of the mechanisms of symbiotic P transport. Mycorrhiza-specific plant phosphate transporters, which are required not only for symbiotic P transfer but also for maintenance of the symbiosis, have been identified. The present review provides an overview of the contribution of AM fungi to plant P acquisition and an update of recent findings on the physiological, molecular and regulatory mechanisms of P transport in the AM symbiosis.

GintAMT3 - a Low-Affinity Ammonium Transporter of the Arbuscular Mycorrhizal Rhizophagus irregularis.

Nutrient acquisition and transfer are essential steps in the arbuscular mycorrhizal (AM) symbiosis, which is formed by the majority of land plants. Mineral nutrients are taken up by AM fungi from the soil and transferred to the plant partner. Within the cortical plant root cells the fungal hyphae form tree-like structures (arbuscules) where the nutrients are released to the plant-fungal interface, i.e., to the periarbuscular space, before being taken up by the plant. In exchange, the AM fungi receive carbohydrates from the plant host. Besides the well-studied uptake of phosphorus (P), the uptake and transfer of nitrogen (N) plays a crucial role in this mutualistic interaction. In the AM fungus Rhizophagus irregularis (formerly called Glomus intraradices), two ammonium transporters (AMT) were previously described, namely GintAMT1 and GintAMT2. Here, we report the identification and characterization of a newly identified R. irregularis AMT, GintAMT3. Phylogenetic analyses revealed high sequence similarity to previously identified AM fungal AMTs and a clear separation from other fungal AMTs. Topological analysis indicated GintAMT3 to be a membrane bound pore forming protein, and GFP tagging showed it to be highly expressed in the intraradical mycelium of a fully established AM symbiosis. Expression of GintAMT3 in yeast successfully complemented the yeast AMT triple deletion mutant (MATa ura3 mep1Δ mep2Δ::LEU2 mep3Δ::KanMX2). GintAMT3 is characterized as a low affinity transport system with an apparent Km of 1.8 mM and a V max of 240 nmol(-1) min(-1) 10(8) cells(-1), which is regulated by substrate concentration and carbon supply.

Deciphering the molecular basis of ammonium uptake and transport in maritime pine.

Ammonium is the predominant form of inorganic nitrogen in the soil of coniferous forests. Despite the ecological and economic importance of conifers, the molecular basis of ammonium uptake and transport in this group of gymnosperms is largely unknown. In this study, we describe the functional characterization of members of the AMT gene family in Pinus pinaster: PpAMT1.1, PpAMT1.2 and PpAMT1.3 (subfamily 1) and PpAMT2.1 and PpAMT2.3 (subfamily 2). Our phylogenetic analysis indicates that in conifers, all members of the AMT1 subfamily evolved from a common ancestor that is evolutionarily related to the ancient PpAMT1.2 gene. Individual AMT genes are developmentally and nutritionally regulated, and their transcripts are specifically distributed in different organs. PpAMT1.3 was predominantly expressed in the roots, particularly during N starvation and mycorrhizal interaction, whereas PpAMT2.3 was preferentially expressed in lateral roots. Immunolocalization studies of roots with varied nitrogen availability revealed that PpAMT1 and PpAMT2 proteins play complementary roles in the uptake of external ammonium. Heterologous expression in yeast and Xenopus oocytes revealed that the AMT genes encode functional transporters with different kinetics and with different capacities for ammonium transport. Our results provide new insights on how nitrogen is acquired and transported in conifers.

A dipeptide transporter from the arbuscular mycorrhizal fungus Rhizophagus irregularis is upregulated in the intraradical phase.

Arbuscular mycorrhizal fungi (AMF), which form an ancient and widespread mutualistic symbiosis with plants, are a crucial but still enigmatic component of the plant micro biome. Nutrient exchange has probably been at the heart of the success of this plant-fungus interaction since the earliest days of plants on land. To characterize genes from the fungal partner involved in nutrient exchange, and presumably important for the functioning of the AM symbiosis, genome-wide transcriptomic data obtained from the AMF Rhizophagus irregularis were exploited. A gene sequence, showing amino acid sequence and transmembrane domains profile similar to members of the PTR2 family of fungal oligopeptide transporters, was identified and called RiPTR2. The functional properties of RiPTR2 were investigated by means of heterologous expression in Saccharomyces cerevisiae mutants defective in either one or both of its di/tripeptide transporter genes PTR2 and DAL5. These assays showed that RiPTR2 can transport dipeptides such as Ala-Leu, Ala-Tyr or Tyr-Ala. From the gene expression analyses it seems that RiPTR2 responds to different environmental clues when the fungus grows inside the root and in the extraradical phase.

Shedding light onto nutrient responses of arbuscular mycorrhizal plants: nutrient interactions may lead to unpredicted outcomes of the symbiosis.

The role and importance of arbuscular mycorrhizae (AM) in plant nitrogen (N) nutrition is uncertain. We propose that this be clarified by using more integrative experimental designs, with the use of a gradient of N supply and the quantification of an extensive array of plant nutrient contents. Using such an experimental design, we investigated AM effects on plant N nutrition, whether the mycorrhizal N response (MNR) determines the mycorrhizal growth response (MGR), and how MNR influences plants' C economy. Oryza sativa plants were inoculated with Rhizophagus irregularis or Funneliformis mossae. AM effects were studied along a gradient of N supplies. Biomass, photosynthesis, nutrient and starch contents, mycorrhizal colonization and OsPT11 gene expression were measured. C investment in fungal growth was estimated. Results showed that, in rice, MGR was dependent on AM nutrient uptake effects, namely on the synergy between N and Zn, and not on C expenditure. The supply of C to the fungus was dependent on the plant's nutrient demand, indicated by high shoot C/N or low %N. We conclude that one of the real reasons for the negative MGR of rice, Zn deficiency of AMF plants, would have remained hidden without an experimental design allowing the observation of plants' response to AM along gradients of nutrient concentrations. Adopting more integrative and comprehensive experimental approaches in mycorrhizal studies seems therefore essential if we are to achieve a true understanding of AM function, namely of the mechanisms of C/N exchange regulation in AM.

Transcriptional regulation of host NH4⁺ transporters and GS/GOGAT pathway in arbuscular mycorrhizal rice roots.

Arbuscular mycorrhizal (AM) fungi play a key role in the nutrition of many land plants. AM roots have two pathways for nutrient uptake, directly through the root epidermis and root hairs and via AM fungal hyphae into root cortical cells, where arbuscules or hyphal coils provide symbiotic interfaces. Recent studies demonstrated that the AM symbiosis modifies the expression of plant transporter genes and that NH4⁺ is the main form of N transported in the symbiosis. The aim of the present work was to get insights into the mycorrhizal N uptake pathway in Oryza sativa by analysing the expression of genes encoding ammonium transporters (AMTs), glutamine synthase (GS) and glutamate synthase (GOGAT) in roots colonized by the AM fungus Rhizophagus irregularis and grown under two N regimes. We found that the AM symbiosis down-regulated OsAMT1;1 and OsAMT1;3 expression at low-N, but not at high-N conditions, and induced, independently of the N status of the plant, a strong up-regulation of OsAMT3;1 expression. The AM-inducible NH4⁺ transporter OsAMT3;1 belongs to the family 2 of plant AMTs and is phylogenetically related to the AM-inducible AMTs of other plant species. Moreover, for the first time we provide evidence of the specific induction of a GOGAT gene upon colonization with an AM fungus. These data suggest that OsAMT3;1 is likely involved in the mycorrhizal N uptake pathway in rice roots and that OsGOGAT2 plays a role in the assimilation of the NH4⁺ supplied via the OsAMT3;1 AM-inducible transporter.

GintAMT2, a new member of the ammonium transporter family in the arbuscular mycorrhizal fungus Glomus intraradices.

In the symbiotic association of plants and arbuscular mycorrhizal (AM) fungi, the fungus delivers mineral nutrients, such as phosphate and nitrogen, to the plant while receiving carbon. Previously, we identified an NH(4)(+) transporter in the AM fungus Glomus intraradices (GintAMT1) involved in NH(4)(+) uptake from the soil when preset at low concentrations. Here, we report the isolation and characterization of a new G. intraradicesNH(4)(+) transporter gene (GintAMT2). Yeast mutant complementation assays showed that GintAMT2 encodes a functional NH(4)(+) transporter. The use of an anti-GintAMT2 polyclonal antibody revealed a plasma membrane location of GintAMT2. GintAMT1 and GintAMT2 were differentially expressed during the fungal life cycle and in response to N. In contrast to GintAMT1, GintAMT2 transcript levels were higher in the intraradical than in the extraradical fungal structures. However, transcripts of both genes were detected in arbuscule-colonized cortical cells. GintAMT1 expression was induced under low N conditions. Constitutive expression of GintAMT2 in N-limiting conditions and transitory induction after N re-supply suggests a role for GintAMT2 to retrieve NH(4)(+) leaked out during fungal metabolism.