Two-way transfer of nitrogen between Dalbergia odorifera and its hemiparasite Santalum album is enhanced when the host is effectively nodulated and fixing nitrogen.

Nutrient translocation from a host plant is vital to the growth and survival of its root parasitic plant, but few studies have investigated whether a parasitic plant is also able to transfer nutrients to its host. The role of N2-fixation in nitrogen (N) transfer between 7-month-old Dalbergia odorifera T. Chen nodulated with Bradyrhizobium elkanii DG and its hemiparasite Santalum album Linn. was examined by external (15)N labeling in a pot study. Four paired treatments were used, with (15)N given to either host or hemiparasite and the host either nodulated or grown on combined N. N2-fixation supplied 41-44% of total N in D. odorifera. Biomass, N and (15)N contents were significantly greater in both nodulated D. odorifera and S. album grown with paired nodulated D. odorifera. Significantly higher total plant (15)N recovery was in N donor D. odorifera (68-72%) than in N donor S. album (42-44%), regardless of the nodulation status in D. odorifera. Nitrogen transfer to S. album was significantly greater (27.8-67.8 mg plant(-1)) than to D. odorifera (2.0-8.9 mg plant(-1)) and 2.4-4.5 times greater in the nodulated pair than in the non-nodulated pair. Irrespective of the nodulation status, S. album was always the N-sink plant. The amount of two-way N transfer was increased by the presence of effective nodules, resulting in a greater net N transfer (22.6 mg plant(-1)) from host D. odorifera to hemiparasite S. album. Our results may provide N management strategies for D. odorifera/S. album mixed plantations in the field.

A role for shoot protein in shoot-root dry matter allocation in higher plants.

BACKGROUND AND AIMS: It is stated in many recent publications that nitrate (NO3-) acts as a signal to regulate dry matter partitioning between the shoot and root of higher plants. Here we challenge this hypothesis and present evidence for the viewpoint that NO3- and other environmental effects on the shoot:root dry weight ratio (S:R) of higher plants are often related mechanistically to changes in shoot protein concentration. METHODS: The literature on environmental effects on S:R is reviewed, focusing on relationships between S:R, growth and leaf NO3- and protein concentrations. A series of experiments carried out to test the proposal that S:R is dependent on shoot protein concentration is highlighted and new data are presented for tobacco (Nicotiana tabacum). KEY RESULTS/EVIDENCE: Results from the literature and new data for tobacco show that S:R and leaf NO3- concentration are not significantly correlated over a range of environmental conditions. A mechanism involving the relative availability of C and N substrates for growth in shoots can explain how shoot protein concentration can influence shoot growth and hence root growth and S:R. Generally, results in the literature are compatible with the hypothesis that macronutrients, water, irradiance and CO2 affect S:R through changes in shoot protein concentration. In detailed studies on several species, including tobacco, a linear regression model incorporating leaf soluble protein concentration and plant dry weight could explain the greater proportion of the variation in S:R within and between treatments over a wide range of conditions. CONCLUSIONS: It is concluded that if NO3- can influence the S:R of higher plants, it does so only over a narrow range of conditions. Evidence is strong that environmental effects on S:R are often related mechanistically to their effects on shoot protein concentration.

The dalbergioid legumes (Fabaceae): delimitation of a pantropical monophyletic clade.

A monophyletic pantropical group of papilionoid legumes, here referred to as the "dalbergioid" legumes, is circumscribed to include all genera previously referred to the tribes Aeschynomeneae and Adesmieae, the subtribe Bryinae of the Desmodieae, and tribe Dalbergieae except Andira, Hymenolobium, Vatairea, and Vataireopsis. This previously undetected group was discovered with phylogenetic analysis of DNA sequences from the chloroplast trnK (including matK) and trnL introns, and the nuclear ribosomal 5.8S and flanking internal transcribed spacers 1 and 2. All dalbergioids belong to one of three well-supported subclades, the Adesmia, Dalbergia, and Pterocarpus clades. The dalbergioid clade and its three main subclades are cryptic in the sense that they are genetically distinct but poorly, if at all, distinguished by nonmolecular data. Traditionally important taxonomic characters, such as arborescent habit, free stamens, and lomented pods, do not provide support for the major clades identified by the molecular analysis. Short shoots, glandular-based trichomes, bilabiate calyces, and aeschynomenoid root nodules, in contrast, are better indicators of relationship at this hierarchical level. The discovery of the dalbergioid clade prompted a re-analysis of root nodule structure and the subsequent finding that the aeschynomenoid root nodule is synapomorphic for the dalbergioids.

Characterisation of rhizobia from African acacias and other tropical woody legumes using Biolog and partial 16S rRNA sequencing.

A Biolog (sole carbon source utilisation) user database of tropical and temperature rhizobial strains was created and used in conjunction with the partial 16S rRNA sequencing method to characterise 12 rhizobial isolates from African acacias and other tropical woody legumes. There was close agreement between the two methods but also some significant discrepancies. A high degree of diversity was shown in the relatively small sample of isolates, with 4 out of 5 of the currently proposed rhizobial genera represented. This is the first time Biolog has shown congruence with genotypic fingerprinting using a wide selection of rhizobial reference and test strains.

An application of NMR microimaging to investigate nitrogen fixing root nodules.

Various techniques to obtain high-resolution NMR images (voxel size down to 39 x 39 x 250 microns) of nitrogen-fixing root nodules from soybean [Glycine max (Merr.)] and peanut (Arachis hypogaea L.) are compared. We describe the artefacts arising from changes in the magnetic susceptibility throughout the sample and how these can be minimised. A series of T1 (TR = 220 to 3020 ms) and T2 (TE = 9.3 to 33.6 ms) weighted images are presented. From these it has been possible to locate the oxygen diffusion barrier. A possible interpretation in terms of nodule biochemistry and physiology are given. The data and parameters presented are shown to serve as a basis for more extensive investigations of root nodules (e.g., the oxygen diffusion barrier or the mechanisms driving the regulation of the oxygen concentration in the infected zone by leghemoglobin) by NMR microimaging.

Natural abundance of 15N and 13C in nodulated legumes and other plants in the cerrado and neighbouring regions of Brazil.

Leaves from over 1000 Brazilian native plants growing in the cerrado and neighbouring regions were sampled for C and N content. Half of these were analysed for 15N and further samples for 13C and ash content. Nodulated legumes from all three sub-families were included, together with two types of reference plant, non-nodulated legumes and non-legumes. Particular emphasis was placed on the large caesalpinioid genus Chamaecrista which is here for the first time reported to fix nitrogen in its native habitats. Woody and herbaceous species of this and other nodulated genera, with the exception of the mimosoid tree Stryphnodendron, showed evidence of nitrogen fixation. Amounts fixed were site-specific as was the 15N signature of reference plants. There was no evidence that nodulated legumes had higher leaf N than non-nodulated legumes: both were higher than non-legumes. Several species of Chamaecrista from section absus and species of Stryphnodendron had carbon contents of 50-55%, higher than previously reported for leaves. This was coupled with low (1-3%) ash contents. The 13C values of plants with ≥49% C were significantly more negative than those with <49% C: most species in the former group were woody and most in the latter group herbaceous. Mimosa pudica was unusual in having a wide range of percent C, percent ash and 13C values; these parameters were significantly correlated. It is concluded that Brazilian native legumes can fix significant amounts of nitrogen in the nutrient-poor cerrado soils. Consideration of mineral and lipid nutrition will be necessary in order fully to understand relations between 13C, carbon content and other physiological parameters.

Stem and root nodules on the tropical wetland legume Aeschynomene fluminensis.

Aeschynomene fluminensis Veil., originally obtained from flooded areas of the Pantanal Matogrossense region of Brazil, was grown under stem-flooded or non-flooded conditions for 70 d after inoculation with isolates of photosynthetic stem nodule rhizobia obtained from native A. fluminensis. Stem nodules formed only on submerged stems of flooded plants (mean of 25 per plant), and did not form on aerial parts, although they were capable of growing and fixing N2 after drainage of the stems. Root nodules formed on both non-flooded and flooded plants but were usually decreased in number by flooding (from means of 124 to 51 per plant, respectively). Flooding (and stem-nodulation) resulted in an increase in shoot (and a decrease in root) dry weight, regardless of rhizobial isolate. Stem nodules were attached by a wide collar of aerenchymatous tissue at the base of the nodule. There were large air spaces in the stem where nodules were subtended and these were continuous with nodule aerenchyma/outer cortex. In addition, aerenchyma and spongy tissue at the base of the nodule connected both flooded and non-flooded root nodules to large intercellular spaces in the root cortex. The stem and root nodules were ovoid in shape, and essentially aeschynomenoid in type, i.e. the central infected tissue was without uninfected, interstitial cells. Root nodules had a similar structure to stem nodules (although stem nodules were generally larger), and flooded root nodules were approximately twice the size of non-flooded nodules. The infected tissue of root and stem nodules consisted of spherical, bacteroid-containing cells containing one or two rod-shaped bacteroids per peribacteroid unit and prominent organelles. Infection threads were observed in root but not in stem nodules. The cortex of stem and root nodules had an apparent oxygen diffusion barrier, consisting of concentric layers of small cells with interlocking cell walls and few intercellular spaces. Cell layers external to these consisted of larger cells and intercellular spaces, with some spaces being occluded with an electron-dense material that contained a glycoprotein recognized by the monoclonal antibodies MAC236 and MAC265. The amount of glycoprotein occlusions did not appear to differ between nodule types or treatments, although stem nodules contained intracellular glycoprotein vesicles adjacent to cell walls. The exterior of the nodules consisted of an epidermis of thin flattened cells with occasional lenticels. Amyloplasts were common in lower stem and hypocotyl nodules, but fewer in flooded or non-flooded root nodules. Upper stem nodules (i.e. those within 6 cm of the water surface) differed from more profoundly submerged stem nodules by having chloroplasts throughout the cortex. Root nodules did not contain chloroplasts, and undifferentiated plastids were found mainly in lower stem nodules.

Molecular separation of genera in Cassiinae (Leguminosae), and analysis of variation in the nodulating species of Chamaecrista.

Randomly Amplified Polymorphic DNA (RAPD) methods have been adapted for use as a phenetic tool on the legume tribe Cassiinae. RAPD-generated polymorphism within local populations was lower than between populations from different geographic regions, between species and genera. Examination of three Cassia species, 12 Chamaecrista species and 13 Senna species using eight primers showed the potential for separation of the nodulated/nitrogen fixing genus Chamaecrista from the previously congeneric groups Cassia and Senna. Similarly, RAPD analysis of two groups of nine Ch. rotundifolia and nine Ch. mimosoides samples using 11 primers has given separation according to both species and to geographical location. Analysis of a small sample of five Chamaecrista species from Brazil with eight primers gave separation consistent with known variations in nodule structure.

Detection of genetic variation between and within populations of Gliricidia sepium and G. maculata using RAPD markers.

Gliricidia sepium and G. maculata are multi-purpose leguminous trees native to Central America and Mexico. Research programmes have been initiated to define the native distribution of Gliricidia and sample the spectrum of genetic variation. To date, there has been little systematic assessment of genetic variability in multi-purpose tree species. Accurate estimates of diversity between- and within-populations are considered a prerequisite for the optimization of sampling and breeding strategies. We have used a PCR-based polymorphic assay procedure (RAPDs) to monitor genetic variability in Gliricidia. Extensive genetic variability was detected between species and the variability was partitioned into between- and within-population components. On average, most (60 per cent) of the variation occurs between G. sepium populations but oligonucleotide primers differed in their capacity to detect variability between and within populations. Population-specific genetic markers were identified. RAPDs provide a cost-effective method for the precise and routine evaluation of variability and may be used to identify areas of maximum diversity. The approaches outlined have general applicability to a range of organisms and are discussed in relation to the exploitation of multi-purpose tree species of the tropics.

The evolutionary significance of the legume genus Chamaecrista, as determined by nodule structure.

Nodulated plants of 14 species of Chamaecrista (Leguminosae, sub-family Caesalpinioideae) were grown. The structure of cells from the active N2 -fixing region of nodules varied greatly with species. At one extreme bacteroids were confined solely by peribacteroid membranes, typical of advanced papilionoid legumes such as soybean. At the other extreme, bacteroids were retained within sheath-bounded persistent infection threads, previously reported as typical of the Caesalpinioideae and also found in certain of the base genera of the Papilionoideae. Other species had an intermediate nodule structure. Nodules of the genus Chamaecrista thus appeared to be at an evolutionary crossroads between the primitive and more advanced forms.

Short-term inhibition of legume N2 fixation by nitrate : I. Nitrate effects on nitrate-reductase activities of bacteroids and nodule cytosol.

The hypothesis of NO2 (-) toxicity as the causative factor of NO3 (-) inhibition of nitrogenase (N2ase; EC 1.18.6.1) activity has been evaluated using a short-term exposure (3 d) of several legumes. Treatment of plants with 10 mM NO3 (-) induced nitrate reductase (NR) from bacteroids (EC 1.7.99.4) and nodule cytosol (EC 1.6.6.1) in most species. Regardless of the levels of both enzymes, significant accumulation of NO2 (-) did not occur in nodules. Dissection of nodules into cortical and infected regions, and subsequent NO2 (-) assays in conditions that suppressed enzyme activities, indicated that, in the short-term, bacteroid NR does not generate NO2 (-) in vivo. This is probably because NO3 (-) access is restricted to the nodule cortex. Accumulation of NO2 (-) at levels that are damaging for N2ase and leghaemoglobin were only observed when a delay occurred between dissection and assaying of nodules. It is concluded that NO2 (-) is not responsible for the initial NO3 (-)-induced decline of N2ase activity, and that toxic amounts of NO2 (-) only build up in nodules following longer exposures to NO3 (-), when this anion is actively reduced by bacteroid and cytosol enzymes.

Short-term inhibition of legume N2 fixation by nitrate : II. Nitrate effects on nodule oxygen diffusion.

A comparison was made of changes in nitrogenase (N2ase; EC 1.18.6.1) activity, oxygen diffusion resistance and NO 3 (-) metabolism in symbioses ofPhaseolus vulgaris L. andVigna radiata (L.) Wilczek during a 3-d exposure to 10 mM NO 3 (-) . Bacteroids fromPhaseolus nodules lacked nitrate reductase (NR;EC 1.7.99.4) but those fromVigna nodules had elevated amounts of the enzyme. The nodule cytosol of both species contained assimilatory NR (EC 1.6.6.1). Both symbioses showed a C2H2-induced decline in N2ase activity, the extent of which remained constant with NO 3 (-) exposure forPhaseolus but became greater forVigna. Nitrate application for 3 d reduced maximum (pre-decline) rates of C2H2-reduction activity by 83% and 36% inPhaseolus andVigna, respectively. Nitrogenase-linked respiration (NLR) closely paralleled N2ase activity as the carbon costs of N2ase were not significantly altered by NO 3 (-) . The relationship between NLR and increases in external O2 concentration from 21 to 60% was used to characterize the oxygen diffusion resistance (R) of nodules from both species. In absolute terms the minimum R ofPhaseolus nodules increased with NO 3 (-) , whereas the ability to adjust this R in response to O2 was lost after 2d. ForVigna nodules the increase in minimum R was much smaller and the adjustment ability was retained for the 3-d period of NO 3 (-) exposure. Bacteroids ofVigna and the cytosol of both species contained NR prior to NO 3 (-) exposure, and activities increased 1.5- to 2-fold during the treatment period. Despite this, NO 2 (-) was not detected in nodules ofPhaseolus, and showed only a very small accumulation in the cytosol ofVigna nodules. It is proposed that nodules have a two-stage response to applied NO 3 (-) . In the first stage NO 3 (-) is restricted to the nodule cortex and causes a reversible increase in R. In the second stage NO 3 (-) may enter the infected region and toxic amounts of NO 2 (-) can be generated in nodules having high bacteroid andor cytosol NR activities. This NO 2 (-) can irreversibly damage the nodules and accelerate their senescence.

Occurrence of nodulation in the Leguminosae.

Reports of nodulation in the Leguminosae are examined in the light of current views on the taxonomy of the family. In the subfamily Caesalpinioideae, nodulation is largely restricted to the tribe Caesalpinieae and the genus Chamaecrista from the Cassieae. All nodules studied have rhizobia retained within infection threads during the nitrogen fixing period. In the Mimosoideae, nodulation is general, except for 4 groups within the tribe Mimoseae, and a very few species of Acacia. The only tribe from the Papilionoideae which appears not to nodulate is the Dipterygeae, although the monogeneric Euchresteae has not been examined. A number of genera in the Swartzieae do not nodulate. Taking tile family as a whole, nodulation appears to be very uniform - certain sections nodulate, others do not.

Which steps are essential for the formation of functional legume nodules?

Nodulation is reviewed in terms of the phenotypes proposed by Vincent (1980). Individual legumes may be infectible by one or more of the three bacterial genera (collectively known as rhizobia) Rhizobium, Bradyrhizobium, or Azorhizobium. The type of infection process by which rhizobia gain entry is largely governed by the host genotype. In addition to the widely studied root-hair pathway, infections may be associated with lateral root emergence or occur between root epidermal cells. The exact chemical and physical nature of the root hair/epidermal cell wall is likely to be a critical factor in determining whether infections can proceed. In addition to differing with species, wall composition may be influenced by soil chemical (e.g. Ca2+ ) and biotic factors (e.g. bacteria). Rhizobial features essential for infection include particular surface polysaccharides and the induction of nodulation genes by plant root exudates. Neither of these is likely to be a major barrier to the extension of nodulation to new hosts. Dissemination of rhizobia within developing nodules may be intercellular, via infection threads or by division of a small number of infected cells. All functional symbioses eventually have 'intracellular' bacteria, in the sense that rhizobia are geographically located within the boundary of the host cell walls. However, they remain extracellular in the sense that they are always confined by a membrane which is largely of host cell origin. In some genera they are also surrounded by infection thread walls, probably modified forms of 'invasive' infection thread walls, which allow differentiation of rhizobia into the nitrogen-fixing form. Thus, natural, functional, symbioses may (a) never involve a stage in which bacteria are confined within tubular infection threads or (b) never release bacteria from infection threads. These features are determined by host genotype. The one feature of legume nodules so far found never to vary is the stem-like character of a peripheral vascular system. This contrasts with the central vascular system of actinorhizas and the rhizobial-induced nodules on the Ulmaceous genus Parasponia. Although of great intrinsic interest, this character is unlikely to present an insurmountable barrier to the extension of nodulation to new species. Other features, such as the ability to produce haemoglobin are now known to the in the genetic makeup of many higher plants. The discovery of the wide range of nodule structures occurring in nature, together with work on mutant rhizobia which may bypass critical stages in the nodulation process, suggest various ways in which the extension of nodulation to non-nodulated legumes and to other (initially at least, dicotyledonous) plants may be engineered. CONTENTS Summary 129 I. Introduction 130 II. The symbionts 130 III. Stages in nodulation 132 IV. Stems and nodules 143 V. Prospects for finding/making new symbioses 144 VI. Conclusions 145 Acknowledgements 147 References 147.

Effects of sodium chloride and polyethylene glycol on root-hair infection and nodulation of Vicia faba L. plants by Rhizobium leguminosarum.

The effects of sodium chloride and polyethylene glycol (PEG) on the interaction between Rhizobium leguminosarum strain 29d and root hairs of field bean (Vicia faba L. cv. Maris Bead) plants were investigated. Two levels each of NaCl (50 and 100 mol·m(-3)) and PEG (100 and 200 mol·m(-3)) were given at the time of root-hair formation. Scanning electron microscopy showed rhizobial attachment and colonization on root-hair tips. Adhesion of rhizobia in both lateral and polar orientation, sometimes associated with microfibrils, occurred mainly in crooks at the root-hair tips; most of the infections also occurred here. Bacterial colonization and root-hair curling were both reduced by stress treatments. Polyethylene glycol but not NaCl significantly reduced root-hair diameter. The proportion of root hairs containing infection threads was reduced by 30% under NaCl and by 52% under PEG. The structure of some of the root hairs, epidermal and hypodermal cells, as seen by light microscopy in ultrasections, was distorted as a result of NaCl and PEG treatments; cells showed plasmolysis and folded membranes. After three weeks of treatment, both NaCl and PEG inhibited nodule number by about 50% and nodule weight by more than 60%. It is concluded that the root-hair infection process in Vicia faba is impaired by NaCl and PEG treatments and this in turn results in fewer nodules being produced.

Calcium-oxalate crystals and crystal cells in determinate root nodules of legumes.

Early reports of the presence of calciumoxalate crystals in the cortices ofPhaseolus vulgaris root nodules have been confirmed. Crystals were found in all six genera examined (Cajanus, Desmodium, Glycine, Lespedeza, Phaseolus, Vigna) that have determinate nodules and export ureides. They were absent from six genera examined that have indeterminate nodules and export amides. The possible physiological significance of these structures is discussed.

Prolonged reduction of acetylene by detached soybean nodules.

Detached soybean nodules reduce acetylene at a constant rate for at least 8 hours provided that (1) only small quantities of nodules per unit volume of gas are used, (2) when incubated in aqueous media they are given sufficient oxygen and shaken, (3) they are not allowed to dry out.

The inability of gibberellic acid to stimulate amylase activity in pea cotyledons.

Gibberellic acid was found to have no significant effect on amylase activity in attached or detached cotyledons of peas.

Effects of benzyladenine on cotyledon metabolism and growth of peas.

Application of benzyladenine to dry pea seed delayed the production of amylase and the concomitant breakdown of starch. The utilization of nitrogenous reserve food material was also delayed. Shoot growth was correlated with breakdown of reserve food.Benzyladenine given to one of the cotyledons did not affect the senescence of the other. Cotyledonary axillary shoots were released from apical dominance, but only those originating from the benzyladenine treated cotyledon continued to develop. The pattern of growth initiated by benzyladenine was not altered by gibberellic acid although this substance caused increased elongation of all growing shoots.