Influence of iron depletion on growth and production of catechol siderophores by different Frankia strains

Nine strains of Frankia isolated from six Casuarinaceae (including four Casuarina sp., one Allocasuarina and one Gymnostoma) and one Elaeagnaceae (Hippophae¨ rhamnoides) were screened for growth and production of siderophores in an iron-deficient liquid medium. Siderophore production was detected only in four strains (Cj, G2, CH and G82) using the CAS and Arnow assays. Salicylates formed more than 90% and dihydroxybenzoates formed less than 10% of all catechol-type siderophores produced. Growth of the former strains was less affected by iron deficiency than that of strains Rif, Thr, URU, BR and RT which do not produce siderophores. Optimal siderophore production by strain Cj was noted when iron concentration reached 0.5μm and was completely inhibited at an iron concentration of 10μm. The kinetics of siderophore production by strain Cj showed that siderophore synthesis was detectable during the growth stationary phase. Growth of Cj (a siderophore-producing strain) and of RT (a non-siderophore-producing strain) differed when 2,2-dipyridyl or ethylene di(o-hydroxyphenyl) acetic acid (EDDHA) was added to the iron-deficient growth medium. Frankia strain RT was the most sensitive to the detrimental effect of both iron chelators.     

Does Ethylene Mediate Cluster Root Formation under Iron Deficiency?

Casuarina glauca develops proteoid (cluster) roots in response to Fe deficiency. This study set out to investigate the possible involvement of ethylene in the initiation and/or the morphogenesis of cluster roots (CR). For this purpose, the effect of Ag+ added as silver thiosulfate, an inhibitor of ethylene action has been studied in plants growing hydroponically. No CR formation was observed in these growth conditions. Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine, 1- aminoisobutyric acid, aminoxyacetic acid or cobalt chloride also eliminated the positive effect of Fe deficiency on CR formation in C. glauca. CR were not formed in Fe- deficient roots in the presence of ethylene inhibitors, suggesting a role for ethylene in the morphological responses to Fe deficiency. Interestingly, treatment of Casuarina plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid stimulated significantly the formation of CR, even if plants are supplied with Fe. However, this stimulation did not reach the level of CR obtained in Fe-deficient plants. These results suggest that an ethylene-mediated signalling pathway is involved in CR formation process in C. glauca.     

Iron deficiency induces cluster (proteoid) root formation in Casuarina glauca

The effect of iron deficiency, phosphorus, NaHCO₃, chelator supply and nitrogen source on the formation of cluster (proteoid) roots was investigated in Casuarina glauca growing in water culture. The addition of iron-binding chelators (e.g. EDDHA, DTPA, EDTA) or increase in nutrient solution pH with NaHCO₃ resulted in the formation of cluster roots when plants were grown in solution lacking iron. Phosphorus supply even at a concentration of 500 µM did not inhibit cluster root formation if EDDHA was added to the iron-deficient medium. Cluster root formation was influenced significantly by nitrogen source and occurred only in nitrate-fed plants.C. glauca seemed to be very sensitive to iron deficiency as shown by plant chlorosis when grown on alkaline soil. The symptoms of chlorosis decreased as the chlorophyll content in shoots and the number of cluster roots increased, suggesting that the alleviation of iron deficiency in plant tissues was correlated with cluster root formation. It appears that iron deficiency is more important than phosphorus deficiency in inducing the formation of cluster roots in C. glauca.     

Is Fe deficiency rather than P deficiency the cause of cluster root formation in Casuarina species?

When subjected, directly (through nutritional deficiencies) or indirectly (through alkaline constraints leading to such deficiencies) to nutrient deficiencies, certain plants respond by developing special root structures called cluster roots. This phenomenon can be considered as an ecophysiological response to a specific nutrient deficiency enabling plants to enhance nutrient uptake. Experiments conducted on an alkaline and an acid soil showed that Casuarina glauca (Sieber ex Spreng.) produced cluster roots only in the alkaline soil and not in the acid soil. In addition, iron (Fe) and phosphorus (P) deficiencies were examined separately or together to determine their effect on cluster root formation in C. glauca seedlings grown hydroponically. Results from experiments carried out on three Casuarina species (C. glauca, C. cunninghamiana Miq. and C. equisetifolia L.) indicated that Fe is involved in cluster root formation. In nutrient media lacking P but containing Fe, no cluster roots formed while seedlings receiving P and lacking Fe developed cluster roots. When incubated on chrome-azurol S-agar on blue plates (CAS assay), a technique used routinely to detect the production of siderophores by micro-organisms, the root system of Fe-deficient plants exhibited orange halos around cluster roots, indicating production of a ferric-chelating agent. It is concluded that the capacity of cluster roots of C. glauca to chelate Fe allows the plant to grow normally on alkaline soils.     

Cluster Roots in Casuarinaceae: Role and Relationship to Soil Nutrient Factors

N₂-fixing actinorhizal trees in the family Casuarinaceae areeconomically of great interest in tropical and sub-tropicalzones because they are used for many purposes including protectionagainst wind, stabilization of sand dunes and the productionof firewood and charcoal. They are usually able to grow in sandysoils with low fertility by virtue of their ability to fix N₂.The objective of this review is to discuss briefly the roleof mycorrhizas and, more extensively, that of cluster (proteoid)roots, developed by a number of species of Casuarinaceae toimprove the absorption of nutrients other than N from soil,especially those needed for N₂fixation and growth. After evaluatingthe actual relationships between mycorrhizas and the Casuarinaceae,we highlight the possible role of cluster roots as an effectivealternative to mycorrhizas, and as a means of improvement ofgrowth of the trees in nutrient-deficient soils. This raisesthe question of what triggers the formation of cluster rootsin the Casuarinaceae. In addition to phosphorus deficiency,iron deficiency seems to be a major factor inducing the formationof cluster roots in Casuarina glauca and C. cunninghamiana.The number of cluster roots and the precocity of their formationare directly related to plant chlorosis due to Fe deficiency,as expressed by the critical concentration of chlorophyll inthe shoot (0.60 mg g ^{- 1}shoot f.wt). The effect of the nitrogensource on cluster root formation is discussed in relation topH values in the plant culture solution. The number of clusterroots formed in nitrate-fed plants increases with pH in therange of 5 to 9. Experiments carried out with alkaline and acidicsoils show that cluster roots are only produced when they areneeded to overcome soil nutrient deficiency due to the immobilizationof nutrient elements (P and Fe) by soil alkalinity. The possibleinvolvement of ethylene in the initiation and/or the morphogenesisof cluster roots is discussed. Copyright 2000 Annals of BotanyCompany Casuarinaceae, Casuarina cunninghamiana, Casuarina glauca, cluster roots, ethylene, iron deficiency, phosphorus deficiency, proteoid roots     

Successful nodulation of Casuarina by Frankia in axenic conditions

AIMS: In order to depict the fine interactions that lead to nodulation, absolute microbiological control of the symbiotic partners is required, i.e. the ability to obtain in vitro axenic nodulation, a condition that has never been fulfilled with the Casuarina-Frankia symbiosis. The effects of culture conditions on plant growth and nodule formation by Casuarina cunninghamiana were investigated. METHODS AND RESULTS: Axenic (capped tubes with different substrates), and nonaxenic cultures (Gibson tubes, pot cultures) were tested. In axenic conditions, C. cunninghamiana, inoculated with Frankia, had poor growth and did not form nodules at 6 weeks. Plants cultivated in Gibson tubes reached the four axillary shoots stage within 6 weeks and formed nodules 4 weeks after inoculation. Sand-pot cultures allowed us to relate the plant development stage at inoculation with nodulation. CONCLUSIONS: The sterile replacement of the cap by a plastic bag increased plant growth and enabled nodule formation 6 weeks after inoculation. The new system of plant culture allows the axenic nodule formation 6 weeks after inoculation. Nodulation behaviour is related to plant development and confinement. SIGNIFICANCE AND IMPACT OF THE STUDY: This axenic plant nodulation system is of major interest in analysing the roles of Frankia genes in nodulation pathways.     

Influence of iron depletion on growth and production of catechol siderophores by different Frankia strains

Nine strains of Frankia isolated from six Casuarinaceae (including four Casuarina sp., one Allocasuarina and one Gymnostoma) and one Elaeagnaceae (Hippophae¨ rhamnoides) were screened for growth and production of siderophores in an iron-deficient liquid medium. Siderophore production was detected only in four strains (Cj, G2, CH and G82) using the CAS and Arnow assays. Salicylates formed more than 90% and dihydroxybenzoates formed less than 10% of all catechol-type siderophores produced. Growth of the former strains was less affected by iron deficiency than that of strains Rif, Thr, URU, BR and RT which do not produce siderophores. Optimal siderophore production by strain Cj was noted when iron concentration reached 0.5m and was completely inhibited at an iron concentration of 10m. The kinetics of siderophore production by strain Cj showed that siderophore synthesis was detectable during the growth stationary phase. Growth of Cj (a siderophore-producing strain) and of RT (a non-siderophore-producing strain) differed when 2,2-dipyridyl or ethylene di(o-hydroxyphenyl) acetic acid (EDDHA) was added to the iron-deficient growth medium. Frankia strain RT was the most sensitive to the detrimental effect of both iron chelators.