Lupinus species differ in their requirements for iron

The effect of iron supply on the growth and nodulation ofLupinus angustifolius L. (Gungurru),Lupinus luteus L. (R-1171) andLupinus pilosus Murr. (P20957) was studied in acid solutions. Plants of the three species were grown together in the same solution and inoculated withBradyrhizobium (Lupinus) WU 425. Plants were then grown with or without applied NH₄NO₃. The lupin species differed greatly in their sensitivity to low iron concentrations in solution withL. pilosus being most tolerant andL. luteus most sensitive.L. pilosus had the highest iron concentration in tissues and had a higher ratio of iron concentration in the youngest fully expanded leaf blades (YEB) to that in roots than the other two species.L. luteus had higher iron concentrations in roots but lower iron concentration in YEB and shoots than didL. angustifolius. The requirements of internal iron for the maximal chlorophyll synthesis in YEB were 65 μg g^-1 forL. angustifolius andL. luteus, and 52 μg g^-1 forL. pilosus. In contrast to effects on growth, the three species had similar external iron requirements for nodule formation in roots and for maximal nitrogen concentrations in shoots. The results indicate that iron tolerant lupin species require lower internal and external iron supply and have a greater ability than sensitive species to translocate iron from roots to shoots.     

High pH in the nutrient solution impairs water uptake in Lupinus angustifolius L.

Root growth in Lupinus angustifolius is greatly decreased when the nutrient solution has a pH above 6.0. This study examined the water relations in this species (cv. Yandee) in response to high pH in solution culture in a glasshouse.The dry weight of roots, the length of taproots and lateral roots and the number of lateral roots were significantly reduced at day 12 after transfer to solution with a pH of 7.5 compared to pH 5.2. This resulted in a marked reduction of total root surface area. However, shoot growth and total leaf area were not affected. In comparison with pH 5.2, plants grown at pH 7.5 in the nutrient solution had a 14–38% more-negative leaf water potential, and their stomatal resistance had increased by 67%.The observations indicate that the impairment of the water relations by high pH is mainly caused by decreased root growth.     

A comparison of structure,development and function in cluster roots of Lupinus albus L. under phosphate and iron stress

Cluster roots are adaptations for nutrient acquisition, found throughout the world in many different plant families and habitats. They arise from changes in root initiation, meristem maintenance and physiology. In Lupinus albus cluster roots form under low internal plant phosphate and low internal plant iron levels. In this study, we compare morphology, structure and physiology of cluster roots formed under –P and –Fe conditions. –Fe cluster roots had a lower density of shorter rootlets than –P roots, and were yellow in colour, probably because of increased phenolics due to down-regulation of peroxidase. Rootlet length and width was reduced in –Fe conditions. The change in exudation of citrate, over time, of –P and –Fe cluster roots shared identical temporal dynamics, with an exudative burst occurring in day 3. However, the –Fe cluster roots displayed much higher rates of exudation than the –P cluster roots. Results are discussed within the context of structural and functional control.     

The development of potential screens based on shoot calcium and iron concentrations for the evaluation of tolerance in Egyptian genotypes of white lupin (Lupinus albus L.) to limed soils

European cultivars of white lupin (Lupinus albus L.) grow poorly in limed or calcareous soils. However, Egyptian genotypes are grown successfully in highly calcareous soil and show no stress symptoms. To examine their physiological responses to alkaline soil and develop potential screens for tolerance, three experiments were conducted in limed and non-limed (neutral pH) soil. Measurements included net CO2 uptake, and the partitioning of Fe2+ and Fe3+ and soluble and insoluble Ca in stem and leaf tissue. Intolerant plants showed clear symptoms of stress, whereas stress in the Egyptian genotypes and in L pilosus Murr. (a tolerant species) was less marked. Only the intolerant plants became chlorotic and this contributed to their reduced net CO2 uptake in the limed soil. In contrast, Egyptian genotypes and L pilosus showed no change in net CO2 uptake between the soils. The partitioning of Ca and Fe either resulted from the stress responses, or was itself a stress response. L pilosus and some Egyptian genotypes differed in soluble Ca concentrations compared with the intolerant cultivars, although no significant difference was apparent in the Ca partitioning of the Egyptian genotype Giza 1. In a limed soil, Giza 1 maintained its stem Fe3+ concentration at a level comparable with that of plants grown in non-limed soil, whereas stem [Fe3+] of an intolerant genotype increased. Gizal increased the percentage of plant Fe that was Fe2+ in its leaf tissue under these conditions; that of the intolerant genotype was reduced. The potential tolerance of the Egyptian genotypes through these mechanisms and the possibility of nutritional-based screens are discussed.     

Nitric oxide is the shared signalling molecule in phosphorus- and iron-deficiency-induced formation of cluster roots in white lupin (Lupinus albus)

Background and Aims

Formation of cluster roots is one of the most specific root adaptations to nutrient deficiency. In white lupin (Lupinus albus), cluster roots can be induced by phosphorus (P) or iron (Fe) deficiency. The aim of the present work was to investigate the potential shared signalling pathway in P- and Fe-deficiency-induced cluster root formation.

Methods

Measurements were made of the internal concentration of nutrients, levels of nitric oxide (NO), citrate exudation and expression of some specific genes under four P × Fe combinations, namely (1) 50 µm P and 10 µm Fe (+P + Fe); (2) 0 P and 10 µm Fe (–P + Fe); (3) 50 µm P and 0 Fe (+P–Fe); and (4) 0 P and 0 Fe (–P–Fe), and these were examined in relation to the formation of cluster roots.

Key Results

The deficiency of P, Fe or both increased the cluster root number and cluster zones. It also enhanced NO accumulation in pericycle cells and rootlet primordia at various stages of cluster root development. The formation of cluster roots and rootlet primordia, together with the expression of LaSCR1 and LaSCR2 which is crucial in cluster root formation, were induced by the exogenous NO donor S-nitrosoglutathione (GSNO) under the +P + Fe condition, but were inhibited by the NO-specific endogenous scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl- 3-oxide (cPTIO) under –P + Fe, +P–Fe and –P–Fe conditions. However, cluster roots induced by an exogenous supply of the NO donor did not secrete citrate, unlike those formed under –P or –Fe conditions.

Conclusions

NO plays an important role in the shared signalling pathway of the P- and Fe-deficiency-induced formation of cluster roots in white lupin.     

The role of copper in nitrogen fixation in Lupinus luteus L.

The effect of copper nutrition on symbiotic N₂ fixation in Lupinus luteus L. was studied. Copper nutrition increased the yield, total nitrogen content and dry weight of nodules. The control plants did not produce pods. Copper deficiency limited iron uptake and its translocation to the nodules. Nodules of copper-deficient plants contained less than half the leghaemoglobin concentration of copper-adequate plants and about one third the polyphenol oxidase activity, tested with catechol as a substrate.     

A high amount of satellite DNA in the genome of Lupinus angustifolius L.

Embryo DNA, isolated from ungerminated seeds of Lupinus angustifolius L., contains an exceptionally high amount of guanine-cytosine-rich satellite DNA. The thermal denaturation curve of total embryo DNA is biphasic with an inflexion point at 62% denaturation, indicating the presence of satellite DNA. The satellite fraction could be separated from the mainband DNA by three successive preparative CsCl-gradient centrifugations. The densities of the DNA fractions are 1.7045 g cm^-3 and 1.6925 g cm^-3, respectively. The percentages of guanine-cytosine calculated from these densities are comparable to the percentages of GC calculated from the melting temperatures. Finally, ressociation studies prove that foldback DNA and highly repeated sequences are much more frequent in the satellite DNA fraction than in the mainband DNA.Abbreviation C _o t the product of the DNA concentration (mol nucleotides l^-1) and the time (s) of incubation in a DNA reassociation reaction - GC guanine-cytosine - np nucleotide parirs - T temperature interval between 16 and 84% denaturation     

Evidence for polynuclear iron(III) clusters in the root nodule bacterium, Rhizobium leguminosarum bv. viciae WSM710

Cells of the root nodule bacterium Rhizobium leguminosarum bv. viciae WSM710 were cultured in a medium containing 20 M ⁵⁷Fe. Mössbauer spectra of the cells at 5.5 and 3.7 K indicated that the major form of iron present in the cells was in the form of polynuclear iron(III) clusters. At 5.5 K the spectral component associated with these clusters was in the form of a superposition of a broad feature (large magnetic hyperfine field distribution) and a doublet. On lowering the temperature of the cells to 3.7 K, the spectral component was transformed into resolved magnetic hyperfine field splitting which yielded a magnetic hyperfine field of 42.4 T when fitted with broad Lorentzian peaks. These spectral characteristics are typical of the hydrated iron(III) phosphate cores of several bacterioferritins. A small fraction (11%) of the Mössbauer spectral area of the cells was in the form of a doublet which yielded parameters ( = 1.35 mm/s; E_Q = 3.15 mm/s) indicative of iron(II). The parameters are very similar to those of a spectral component previously observed in several other microbes (R. Böhnke and B.F. Matzanke (1995) BioMetals 8, 223-230) and which has been associated with a 2.2 kDa oligomeric iron(II) carbohydrate phosphate     

Microvirga tunisiensis sp. nov., a root nodule symbiotic bacterium isolated from Lupinus micranthus and L. luteus grown in Northern Tunisia

Three bacterial strains, LmiM8^T, LmiE10 and LluTb3, isolated from nitrogen-fixing nodules of Lupinus micranthus (Lmi strains) and L. luteus (Llu strain) growing in Northern Tunisia were analysed using genetic, phenotypic and symbiotic approaches. Phylogenetic analyses based on rrs and concatenated gyrB and dnaK genes suggested that these Lupinus strains constitute a new Microvirga species with identities ranging from 95 to 83% to its closest relatives Microvirga makkahensis, M. vignae, M. zambiensis, M. ossetica, and M. lotononidis. The genome sequences of strains LmiM8^T and LmiE10 exhibited pairwise Average Nucleotide Identities (ANIb) above 99.5%, significantly distant (73–89% pairwise ANIb) from other Microvirga species sequenced (M. zambiensis and M. ossetica). A phylogenetic analysis based on the symbiosis-related gene nodA placed the sequences of the new species in a divergent clade close to Mesorhizobium, Microvirga and Bradyrhizobium strains, suggesting that the M. tunisiensis strains represent a new symbiovar different from the Bradyrhizobium symbiovars defined to date. In contrast, the phylogeny derived from another symbiosis-related gene, nifH, reproduced the housekeeping genes phylogenies. The study of morphological, phenotypical and physiological features, including cellular fatty acid composition of the novel isolates demonstrated their unique profile regarding close reference Microvirga strains. Strains LmiM8^T, LmiE10 and LluTb3 were able to nodulate several Lupinus spp. Based on genetic, genomic and phenotypic data presented in this study, these strains should be grouped within a new species for which the name Microvirga tunisiensis sp. nov. is proposed (type strain LmiM8^T = CECT 9163^T, LMG 29689^T).     

Interactions Between High pH and Iron Supply on Nodulation and Iron Nutrition of Lupinus albus L. Genotypes Differing in Sensitivity to Iron Deficiency

Poor growth of white lupin (Lupinus albus L.) on alkaline soils may result from its sensitivity to iron deficiency and poor nodulation. This study examined interactive effects of iron supply and high pH on the growth and nodulation of three genotypes differing in their sensitivity to iron deficiency. Three genotypes (P27486, Ultra and WTD180) were grown for 17 days in buffered solutions with Fe supply of 0.2, 2 and 20 μM. Solution pH was adjusted to 5.2, 6.5 or 7.5. Plant growth, nodulation and nutrient concentrations in plants were measured. Decreasing Fe supply decreased chlorophyll concentration in young leaves by up to 92%. Increasing pH decreased chlorophyll concentration by an average of 40% at pH 6.5 and by 47% at pH 7.5. The decrease of chlorophyll was less obvious in P27485 than in Ultra or WTD180. Shoot biomass was reduced by up to 18% by Fe deficiency, with such decrease being less for P27486. Increasing pH exacerbated the effect of Fe deficiency on shoot biomass only of Ultra. Decreasing Fe supply decreased nodule number by an average of 54%, and increasing pH decreased nodule number by 80%. P27486 formed the greatest number of nodules while WTD180 the least. P27486 had high Fe uptake and low internal requirement. Irrespective of genotype, leaf chlorosis positively correlated with cluster root formation. The results suggest that a combination of Fe deficiency and high pH impaired nodulation in L. albus, and that selection of genotypes for both tolerance of iron deficiency and good nodulation at high pH is important for a successful lupin crop on alkaline soils.     

A strategy to develop molecular markers applicable to a wide range of crosses for marker assisted selection in plant breeding: a case study on anthracnose disease resistance in lupin (Lupinus angustifolius L.)

A key challenge in marker-assisted selection (MAS) for molecular plant breeding is to develop markers linked to genes of interest which are applicable to multiple breeding populations. In this study representative F₂ plants from a cross Mandalup (resistant to anthracnose disease) × Quilinock (susceptible) of Lupinus angustifolius were used in DNA fingerprinting by Microsatellite-anchored Fragment Length Polymorphism (MFLP). Nine candidate MFLP markers linked to anthracnose resistance were identified, then ‘validated’ on 17 commercial cultivars. The number of “false positives” (showing resistant-allele band but lack of the R gene) for each of the nine candidate MFLP markers on the 17 cultivars ranged from 1 to 9. The candidate marker with least number of false positive was selected, sequenced, and was converted into a co-dominant, sequence-specific, simple PCR based marker suitable for routine implementation. Testing on 180 F₂ plants confirmed that the converted marker was linked to the R gene at 5.1 centiMorgan. The banding pattern of the converted marker was consistent with the disease phenotype on 23 out of the 24 cultivars. This marker, designated “AnManM1”, is now being used for MAS in the Australian lupin breeding program. We conclude that generation of multiple candidate markers, followed by a validation step to select the best marker before conversion to an implementable form is an efficient strategy to ensure wide applicability for MAS.     

Effects of low molybdenum seed on nodule initiation,development and N2 fixation in black gram (Vigna mungo L.)

In legumes, both increases and decreases in nodule number in response to Mo deficiency have been reported, but reasons for the different responses have not been proposed. The present study examined nodule initiation and development in black gram seedlings using two levels of seed Mo to induce Mo deficiency. In the first 11 days after inoculation, low levels of Mo in seed had no effect on nodule initiation or the number of nodules. At 13 days after inoculation, low Mo in seed depressed bacteroid concentration, leghaemoglobin concentration, nodule number and nodule fresh weight. Acetylene reduction activity was delayed by 2 days in plants grown from low Mo seed. We suggest that the delay in N₂ fixation in plants grown from low Mo seed was due to slower incorporation of Mo of soil origin into nitrogenase. We further suggest that restricted supply of essential metabolites to the nodules on plants from low Mo seed resulted in the slower maturation of early initiated nodules and the repression of formation of new nodules.     

Nitric oxide and glutathione impact the expression of iron uptake- and iron transport-related genes as well as the content of metals in A. thaliana plants grown under iron deficiency

Mounting evidence indicate that nitric oxide (NO) acts as a signaling molecule mediating iron deficiency responses through the upregulation of the expression of iron uptake-related genes. Accordingly, NO donors such as nitrosoglutathione (GSNO) were reported to improve the fitness of plants grown under iron deficiency. Here, we showed that glutathione, a by-product of GSNO, triggered the upregulation of the expression of iron uptake- and transport-related gene and an increase of iron concentration in Arabidopsis thaliana seedlings facing iron deficiency. Furthermore, we provided evidence that under iron deficiency, NO released by GSNO did not improve the root iron concentration but impacted the content of copper. Collectively, our data highlight the complexity of interpreting data based on the use of NO donors when investigating the role of NO in iron homeostasis.