Melanin biosynthesis by Frankia strain CeI5.

Many Frankia strains are pigmented and presumed to produce melanin. However, melanin biosynthesis has yet to be rigorously characterized in Frankia. This study was initiated to determine whether or not Frankia strain CeI5 produced melanin and to identify the biochemical pathway of pigment production. Frankia strain CeI5 first produced a dark pigment in mycelial and other tissue and then in the liquid culture medium when grown in a defined medium containing l-tyrosine. The pigment resisted solvents, lightened when subjected to the action of oxidants, as well as reductants, and produced a flocculent brown precipitate with FeCl(3). Spectroscopic characteristics of the extracted pigment were those of melanin. When subjected to gradual dilution, the absorbance decreased unevenly, occurring in the near red range first, then in the visible range, and lastly in the UV range. This observation might resolve the question of why quite different descriptions of melanin UV-visible light absorption spectra exist in the literature. The tyrosinase cofactor copper greatly enhanced melanin biosynthesis at 5.3 x 10(-6) M, while 1 x 10(-8) M 3,4-dihydroxy-l-phenylalanine hastened pigmentation. The copper-chelating agent KCN and the tyrosinase inhibitor tropolone decreased melanin production at the same concentration of 1 x 10(-5) M. This evidence suggests that Frankia strain CeI5 produces melanin via the Raper and Mason pathway.

Effect of P availability on temporal dynamics of carbon allocation and glomus intraradices high-affinity P transporter gene induction in arbuscular mycorrhiza.

Arbuscular mycorrhizal (AM) fungi depend on a C supply from the plant host and simultaneously provide phosphorus to the colonized plant. We therefore evaluated the influence of external P on C allocation in monoxenic Daucus carota-Glomus intraradices cultures in an AM symbiosis. Fungal hyphae proliferated from a solid minimal medium containing colonized roots into a C-free liquid minimal medium with high or low P availability. Roots and hyphae were harvested periodically, and the flow of C from roots to fungus was measured by isotope labeling. We also measured induction of a G. intraradices high-affinity P transporter to estimate fungal P demand. The prevailing hypothesis is that high P availability reduces mycorrhizal fungal growth, but we found that C flow to the fungus was initially highest at the high P level. Only at later harvests, after 100 days of in vitro culture, were C flow and fungal growth limited at high P availability. Thus, AM fungi can benefit initially from P-enriched environments in terms of plant C allocation. As expected, the P transporter induction was significantly greater at low P availability and greatest in very young mycelia. We found no direct link between C flow to the fungus and the P transporter transcription level, which indicates that a good C supply is not essential for induction of the high-affinity P transporter. We describe a mechanism by which P regulates symbiotic C allocation, and we discuss how this mechanism may have evolved in a competitive environment.

The influence of external nitrogen on carbon allocation to Glomus intraradices in monoxenic arbuscular mycorrhiza.

The influence of external nitrogen (N) on carbon (C) allocation and processes related to phosphorus (P) metabolism were studied in monoxenic arbuscular mycorrhiza (AM) cultures of Daucus carota. Fungal hyphae of Glomus intraradices proliferated from colonized roots growing on solid medium into C-free liquid minimal medium with two different N and P levels. Furthermore, we exposed the colonized roots to high or low N availability and then studied the mycelial development. Roots were provided with (13)C-glucose in order to follow the C allocation. The mycelium was analysed for phosphatase activity and transcription levels of two nutrient regulated genes. High N availability to the monoxenic AM root reduced the C allocation to the AM fungus while N availability to the mycelium was important for the upregulation of the fungal inorganic phosphorus (Pi)-transporter GiPT. We found that N availability can regulate nutritional processes in arbuscular mycorrhiza. We conclude that negative impacts of N on AM abundance are caused by reduced C allocation from the plant. Upregulation of the fungal Pi-transporter GiPT indicated that increased N availability might induce P limitation in the mycelium.

A plasma membrane zinc transporter from Medicago truncatula is up-regulated in roots by Zn fertilization, yet down-regulated by arbuscular mycorrhizal colonization.

Here we present a Zn transporter cDNA named MtZIP2 from the model legume Medicago truncatula. MtZIP2 encodes a putative 37 kDa protein with 8-membrane spanning domains and has moderate amino acid identity with the Arabidopsis thaliana Zn transporter AtZIP2p. MtZIP2 complemented a Zn-uptake mutant of yeast implying that the protein encoded by this gene can transport Zn across the yeast's plasma membrane. The product of a MtZIP2-GFP fusion construct introduced into onion cells by particle bombardment likewise localized to the plasma membrane. The MtZIP2 gene was expressed in roots and stems, but not in leaves of M. truncatula and, in contrast to all other plant Zn transporters characterized thus far, MtZIP2 was up-regulated in roots by Zn fertilization. Expression was highest in roots exposed to a toxic level of Zn. MtZIP2 expression was also examined in the roots of M. truncatula when colonized by the obligate plant symbiont, arbuscular mycorrhizal (AM) fungi, since AM fungi are renowned for their ability to supply plants with mineral nutrients, including Zn. Expression was down-regulated in the roots of the mycorrhizal plants and was associated with a reduced level of Zn within the host plant tissues.

Functional diversity of arbuscular mycorrhizas extends to the expression of plant genes involved in P nutrition.

This study of functional diversity considers symbiotic associations between two plant species, Medicago truncatula and Lycopersicon esculentum, and seven species of arbuscular mycorrhizal fungi (AMF). The objective was to integrate physiological analyses with molecular techniques to test whether functional diversity between AMF species is not only apparent at the level of mycorrhiza formation, plant nutrient uptake and plant growth, but also at the molecular level as observed by variation in the root expression of plant genes involved in the plant's P-starvation response. The seven species of AMF varied widely in their influence on the root expression of MtPT2 and Mt4 from M. truncatula and LePT1 and TPSI1 from L. esculentum. At one extreme was Glomus mosseae, whereby its colonization of M. truncatula resulted in the greatest reduction in MtPT2 and Mt4 gene expression and the highest level of P uptake and growth, while at the other extreme was Gigaspora rosea, whereby colonization resulted in the highest levels of MtPT2 and Mt4 gene expression and the lowest P uptake and growth. The expression of LePT1 and TPSI1 within the roots of L. esculentum was low and relatively uniform across the seven mycorrhizas, reflecting the ability of this cultivar to maintain low and constant shoot P levels despite root colonization by a broad selection of AMF. This study extends current understanding of functional diversity and shows that plants can respond differently to AMF, not only at the level of colonization, nutrient uptake and growth, but also at the level of gene expression.