Modelling the flow and partitioning of carbon and nitrogen in the holoparasite Cuscuta reflexa Roxb. and its host Lupinus albus L.: I. Methods for estimating net flows

Nodulated Lupinus albus L. was grown on quartz sand in the greenhouseand supplied with a N-free culture solution. Half the plantswere infected with Cuscuta reflexa Roxb. at 33 DAS. An empiricallybased modelling technique was developed to quantitatively depictuptake, flow and utilization of C and N in the host plant andbetween host and parasite over a 12 d period. The modellingincorporated C: N ratios of solutes in phloem and pressure-inducedxylem sap, net increments of C and N and respiratory lossesof C. For assessing the transfer of solutes from host phloemto Cuscuta it was not possible to use the C: N ratio of phloemsap close to the site of parasite attachment, a procedure whichwould have assumed non-specific withdrawal of phloem-borne solutes,since this would have implied unimpeded mass flow from hostto parasite. The relative intake of C and N by the parasiteby specific withdrawal of nitrogenous and carbonaceous solutesfrom the phloem was obtained independently by assuming thatxylem intake occurred non-specifically. Xylem import was thusobtained (a) from transpiration and tissue water increment ofCuscuta and the concentrations of N and C in xylem sap and (b)from the Ca²⁺ increment of Cuscuta and the ratios Ca: N andCa: C in lupin xylem sap, assuming that Ca²⁺ intake occurredsolely via xylem. By subtracting net xylem import from totaluptake of C and N by Cuscuta the methods resulted in comparableratios of C: N intake from the phloem. The average ratio (53.4)was smaller than the C:N ratio in host phloem (85.6) indicatingspecific withdrawal of solutes with a distinct preference forN. Using this ratio, modelling of flows of C and N was possibleand showed that Cuscuta abstracted C and N mainly from the hostphloem, but xylem supply was nutrient-dependent and amountedto 6.4% of the N but only 0.5% of the C demand. The resultsindicated that Cuscuta exerted a very strong sink and competedefficiently with the root, the major sink of L. albus, by attracting81% of the current photosynthate and more N (223%) than wascurrently fixed. The massive demand of the parasite led to lossesparticularly of N from leaves and the root and apart from causingcarbon losses it appeared to induce a sink-dependent stimulationof photosynthesis. In contrast, nitrogen fixation in the Cuscuta-infectedlupin was inhibited to 37% of the control. Key words: Cuscuta reflexa, Lupinus albus, carbon, nitrogen, phloem, xylem, transport, parasites, modelling     

Modelling of quinolizidine alkaloid net flows in Lupinus albus and between L. albus and the parasite Cuscuta reflexa: new insights into the site of quinolizidine alkaloid synthesis

The present work reveals new and completely different conclusionsabout the alkaloid economy of symbiotically fed Lupinus albusand L. albus parasitized by Cuscuta reflexa in the study periodof 43–55 d after sowing of lupin. Net flows of alkaloidswithin lupin and between host and parasite were calculated usingthe molar ratio of alkaloid nitrogen: total nitrogen combinedwith known net flows of nitrogen in the transport fluids andanalysing alkaloid accumulation in plant organs by HRGC. Incontrast to previous studies, quinolizidine alkaloids were predictedto be synthesized mainly in the root of L. albus and to be predominantlytransported via xylem to the apical plant shoot organs. Parasitismby C. reflexa for 12 d induced a decline of alkaloid contentin the host L. albus up to 53% compared to control plants andalkaloid synthesis was halved—apparently due to a shortageof the precursor lysine. In spite of an additional decreasein nitrogen levels at the second harvest, the host-parasitesystem showed a1.3-fold higher alkaloid content than the controlplants, 63% of the total alkaloids being attracted by Cuscuta.This indicates (a) restriction of catabolic processes withininfected lupins, (b) a massive shift of nitrogen metabolismin the direction of alkaloids and (c) an enormous sink potentialof Cuscuta for nitrogenous compounds. Although xylem was foundto be the main translocation system for alkaloids, the modellingof alkaloid flows predicts Cuscuta to derive only 4.5% of itstotal alkaloid supply from the xylem and 95.5% from the phloem.By analogy with nitrogen flows, this finding requires xylemphloemtransfers which were assumed to occur within the stem axis oflupin. A similar proportion regarding the contribution of xylemand phloem to the supply of Cuscuta was obtained for the netflows of two selected alkaloids, lupanine and 13     

Studies on Cuscuta reflexa ROXB. V. The level of endogenous hormones in the parasite,Cuscuta reflexa,and its host,Vicia faba L., and a suggested role in the transfer of nutrients from host to parasite

Summary The mechanism of parasitism of Cuscuta, especially the absorption of nutrients from its host, is not clear. As it might be connected with the function of plant hormones, the endogenous levels of all hormone groups in the parasite, Cuscuta reflexa, and its host, Vicia faba, were investigated. Since the content of auxins, gibberellins and cytokinins is higher in the host than in the parasite, there is no indication that any of these phytohormones is involved in the absorption of nutrients by Cuscuta. However, the content of growth inhibitors, especially free abscisic acid, is much higher in Cuscuta than in the host. There is a gradient of abscisic acid with a maximum in the basal, haustoria-bearing stem region in which the transfer of nutrients from host to parasite occurs. The high content of abscisic acid within the parasite may be a causal connection with the parasitic absorption of nutrients from host sieve tubes.This publication is respectfully dedicated to the academician Prof. Dr. A. L. Kursanov, Moscow, on the occasion of the 80th anniversary of his birthday.     

Alterations in carbon and nitrogen metabolism induced by water deficit in the stems and leaves of Lupinus albus L.

Water deficit (WD) in Lupinus albus L. brings about tissue-specific responses that are dependent on stress intensity. Carbohydrate metabolism is very sensitive to changes in plant water status. Six days from withholding water (DAW), sucrose, glucose and fructose levels of the leaf blade had already increased over 5-fold, and the activities of SS and INV(A) had increased c. 1.5-2 times. From 9 DAW on, when stress intensity was more pronounced, these effects were reversed with fructose and glucose concentrations as well as INV(A) activity dropping in parallel. The stem (specifically the stele) responded to the stress intensification with striking increases in the concentration of sugars, N and S, and in the induction of thaumatin-like-protein and an increase in chitinase and peroxidase. At 13 DAW, the plants lost most of the leaves but on rewatering they fully recovered. Thus, the observed changes appear to contribute to a general mechanism of survival under drought, the stem playing a key role in that process.     

Interaction of a host plant and its holoparasite: effects of previous selection by the parasite

If parasites decrease the fitness of their hosts one could expect selection for host traits (e.g. resistance and tolerance) that decrease the negative effects of parasitic infection. To study selection caused by parasitism, we used a novel study system: we grew host plants (Urtica dioica) that originated from previously parasitized and unparasitized natural populations (four of each) with or without a holoparasitic plant (Cuscuta europaea). Infectivity of the parasite (i.e. qualitative resistance of the host) did not differ between the two host types. Parasites grown with hosts from parasitized populations had lower performance than parasites grown with hosts from unparasitized populations, indicating host resistance in terms of parasite’s performance (i.e. quantitative resistance). However, our results suggest that the tolerance of parasitic infection was lower in hosts from parasitized populations compared with hosts from unparasitized populations as indicated by the lower above‐ground vegetative biomass of the infected host plants from previously parasitized populations.     

Mechanism of parasitization by Cuscuta reflexa: RNase activity in the haustorial region of Cuscuta and infected host tissues

In a study of host-parasite interrelationship and the mechanism of parasitization, ribonuclease activity was determined in Cuscuta reflexa Roxb, and in infected and control host plants of Lantana camara L. In the haustoria-bearing region of Cuscuta , the concave half of the vine bearing the haustorial site, always showed significantly higher RNase activity than the convex half, irrespective of the differing enzyme activity of the parasite tissue growing on different hosts — Brassica campestris L., Helianthus animus L., Lantana camara L., Medicago saliva L. and Solatium nigrum L. The uninfected host branch of L. camara showed the maximum specific RNase activity in the apical region which decreased toward the base, while the infected host branch showed minimum specific RNase activity in the apical region, gradually increasing towards the infected region.     

Influence of the obligate parasite Cuscuta campestris on growth and biomass allocation of its host Mikania micrantha

As a means of biologically controlling Mikania micrantha H.B.K. in South China, the influence of the obligate parasite Cuscuta campestris Yuncker on its growth and biomass allocation was studied using pot trials. The effect of C. campestris on M. micrantha became greater with time, such that the host biomass was only 1.8% of the control after 60 d of parasitism and by day 72 almost all the aerial parts of the host plants had died. Afterwards, the hosts and the remnant parasite shoots re-grew but the total biomass of the hosts was still significantly lower than that of the controls. The infection by C. campestris greatly increased the shoot:root dry weight ratio and the allocation to stems of the infected plants from 40 to 50 d after parasitization, but decreased their relative growth rate and unit leaf rate starting from 20 d after parasitization and their leaf area ratio from 30 to 60 d after parasitization. Cuscuta campestris significantly reduced the total biomass, changed the biomass allocation patterns, and completely inhibited the flowering of the infected M. micrantha plants. These results indicate that the use of C. campestris could be a potentially effective way of controlling M. micrantha.     

Influence of the obligate parasite Cuscuta campestris on growth and biomass allocation of its host Mikania micrantha

Vol. 56, No. 415, May     

Lateral diffusion of polarly transported indoleacetic acid and its role in the growth of Lupinus albus L. hypocotyls

The transport and metabolism of indole-3-acetic acid (IAA) was studied in etiolated lupin (Lupinus albus L, cv. Multolupa) hypocotyls, following application of dual-isotope-labelled indole-3-acetic acid, [5-³H]IAA plus [1-¹⁴C]IAA, to decapitated plants. To study the radial distribution of the transported and metabolized IAA, experiments were carried out with plants in which the stele was separated from the cortex by a glass capillary. After local application of labelled IAA to the cortex, radioactivity remained immobilized in the cortex, near the application point, showing that polar transport cannot occur in the outer tissues. However, following application of IAA to the stele, radioactivity appeared in the cortex in those hypocotyl sections below the first 1 cm (in which the capillary was inserted), and the basipetal IAA movement was similar to that observed after application of IAA to the complete cut surface. In both assays, longitudinal distribution of ¹⁴C and ³H in the stele outside the first 1 cm was positively correlated with that of cortex, indicating that there was a lateral migration of IAA from the transport pathway (in the stele) to the outer tissues and that this migration depended on the amount of IAA in the stele. Both tissues (stele and cortex) exhibited intensive IAA metabolism, decarboxylation being higher in the stele than in the cortex while IAA conjugation was the opposite. Decapitation of the seedlings caused a drastic reduction of hypocotyl growth in the 24 h following decapitation, unless the hypocotyls were treated apically with IAA. Thus, exogenous IAA, polarly transported, was able to substitute the endogenous source of auxin (cotyledons plus meristem) to permit hypocotyl growth. It is proposed that IAA escapes from the transporting cells (in the stele) to the outer tissues in order to reach the growth-responsive cells. The IAA metabolism in the outer tissues could generate the IAA gradient necessary for the maintenance of its lateral flow, and consequently the auxin-induced cell elongation.     

Effect of high external NaCl concentration on ion transport within the shoot of Lupinus albus. II. Ions in phloem sap

Abstract. Phloem sap was collected from petioles of growing and fully expanded leaves of lupins exposed to 0–150 mol m⁻³ [NaCl]_ext, for various periods of time. Sap bled from growing leaves only after the turgor of the shoot was raised by applying pneumatic pressure to the root. Increased pressure was also needed to obtain sap from fully expanded leaves of plants at high [NaCl]_ext. Exposure to NaCl caused a rapid rise in the Na⁺ concentration in phloem sap to high levels. The Na⁺ concentration reached 20 mol m⁻³ within a day of exposure and reached a plateau of about 60 mol m⁻³ in plants at 50–150 mol m⁻³ [NaCl]_ext, after a week. There was a slower, smaller increase in the Cl⁻ concentration. K⁺ concentrations in phloem sap were not affected by [NaCl]_ext. Cl⁻ concentrations in phloem sap collected from growing leaves were similar to those from old leaves while Na⁺ concentrations were somewhat increased, suggesting that there was no reduction in the salt content of the phloem sap while it flowed within the shoot to the apex. Calculations of ion fluxes in xylem and phloem sap indicated that Na⁺ and Cl⁻ fluxes in the phloem from leaves of plants at high NaCl could be equal to those in the xylem. This prediction was borne out by observations that Na⁺ and Cl⁻ concentrations in recently expanded leaves remained constant.     

Modelling partitioning between structure and storage in sugar beet: Effects of drought and soil nitrogen

In the UK sugar beet is grown on contrasting soils that vary both in their nutritional status and water-holding capacities. Water shortage and low nitrogen reduce canopy growth and dry matter production, which is compensated in part by an increase in the fraction of assimilates partitioned to storage. Conversely, high nitrogen and ample water encourage growth of the canopy, increase assimilation of carbon dioxide but reduce the proportion of assimilates stored as sugar. This paper sets out to examine simple relationships between sugar yield, total dry matter and soil nitrogen in rain-fed and irrigated sugar beet crops (Beta vulgaris L.) from 46 field experiments spanning 12 years and a range of soil types, in order to improve prediction of sugar yields. Two partitioning functions were fitted to the data. The first represents a useful alternative formulation of the allometric growth function that overcomes some of the difficulties in the interpretation of the parameters. This model adequately described the seasonal progress of sugar yield (Y) in relation to total dry matter (W), but it was difficult to postulate biological mechanisms as to how the parameters should vary in relation to varying soil nitrogen or to drought. The second partitioning function, given by Y = W − (1/k) log(1 + kW), also described the data well, but had the more useful parameter, k, the decay rate of the fraction of assimilates partitioned to structural matter. This was shown to be greater in crops which had experienced significant drought and was inversely proportional to the amount of nitrogen taken up by the crops. Relationships between k and amounts of nitrogen fertilizer applied and/or amounts of residual nitrogen in the soil at sowing, however, were more variable. These could be improved by additionally taking account of soil type and rainfall following nitrogen fertilizer application in late spring. The models are a useful extension to yield forecasting models because they provide a simple means of estimating sugar yield from total dry matter in relation to factors that affect partitioning of assimilates such as drought and soil nitrogen availability. This revised version was published online in June 2006 with corrections to the Cover Date.     

Investigations into the exploitation of heterogeneous soils by Lupinus albus L. and L. pilosus Murr. and the effect upon plant growth

In calcareous soils, genotypes of Lupinus albus L. generally grow poorly, resulting in stunted plants that often develop lime-induced chlorosis. In contrast, some genotypes of L. pilosus Murr. occur naturally in calcareous soils without developing any visible symptoms of stress. Some genotypic variation for tolerance to calcareous soil does exist in L. albus and the tolerance mechanisms need to be determined. The adaptation through root system morphological plasticity of L. albus and L. pilosus, to heterogeneous limed soil profiles (pH 7.8) containing either patches of acid (non-limed) soil, or vertically split between acid and limed soil, was investigated. When grown in the presence of patches of acid soil, L. albus had a 52% greater shoot dry weight and visibly greener leaves compared with plants grown in the homogeneous limed soil. Total root dry matter in the acid-soil patches was greater than in the control limed-soil patches. This was due to a four-fold increase in the cluster root mass, accounting for 95% of the root dry matter in the acid-soil patch. Although these cluster roots secreted no more citric acid per unit mass than those in the limed soil did, their greater mass resulted in a higher citrate concentration in the surrounding soil. L. pilosus responded to the patches of acid soil in a manner comparable with L. albus. When grown in the homogeneous limed soil, L. pilosus had a greater maximum net CO₂ assimilation rate (P_max) than L. albus, however, the P_max of both species increased after they had accessed a patch of acid soil. Differences were apparent between the L. albus genotypes grown in soil profiles split vertically into limed and acid soil. A genotype by soil interaction occurred in the partitioning between soils of the cluster roots. The genotype La 674 was comparable with L. pilosus and produced over 11% of its cluster roots in the limed soil, whereas the other genotypes produced only 1–3% of their cluster roots in the limed soil. These results indicate L. pilosus is better adapted to the limed soil than L. albus, but that both species respond to a heterogeneous soil by producing mainly cluster roots in an acid-soil patch. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phylogenetic and symbiotic diversity of Lupinus albus and L. angustifolius microsymbionts in the Maamora forest,Morocco

Out of 70 bacterial strains isolated from root nodules of Lupinus albus and L. angustifolius grown in the soils from the Maamora forest in Morocco, 56 isolates possessed the nodC symbiotic gene, as determined by nodC-PCR, and they were able to renodulate their original hosts.The phenotypic analysis showed that many strains had great potential for using different carbon compounds and amino acids as sole carbon and nitrogen sources. The majority of strains grew in media with pH values between 6 and 8. Only one strain isolated from L. angustifolius was able to grow at low pH values, whereas fourteen strains nodulating L. albus grew at pH 5. No strain developed at 40 °C, and eighteen strains grew at NaCl concentrations as high as 855 mM. A total of 17 strains solubilized phosphates, whereas 20 produced siderophores and seven produced IAA. Only three strains, Lalb41, Lang10 and Lang16, possessed all three plant growth promoting activities. The strains were grouped into eight genetic groups by rep-PCR. Analysis of the 16S rRNA sequences of eight strains representing the different groups showed that they were members of the genus Bradyrhizobium. The sequencing of the five housekeeping genes atpD, glnII, dnaK, gyrB and recA, from the eight representative strains, and the phylogenetic analysis of their concatenated sequences, showed that both plants were nodulated by different Bradyrhizobium species. Accordingly, two strains, Lalb41 and Lalb5.2, belonged to B. lupini, whereas two strains, Lalb2 and Lang17.2, were affiliated to B. cytisi, and one strain, Lang2, was close to B. canariense. The fourth group of strains, Lalb25, Lang14.3 and Lang8.3, which had similarity values of less than 96% with their closest named species, B. cytisi, may belong to two new genospecies in the genus Bradyrhizobium. All the strains nodulated Lupinus cosentinii, L. luteus, Retama sphaerocarpa, R. monosperma, Chamaecytisus albus, but not Vachellia gummifera, Phaseolus vulgaris or Glycine max. The nodA, nodC and nifH sequence analyses and their phylogeny confirmed that the strains isolated from the two lupines were members of the symbiovar genistearum.     

Spatial and energy compromise between host and parasite: the Biomphalaria glabrata-Schistosoma mansoni system.

The development of a sporocyst infrapopulation of Schistosoma mansoni within the Biomphalaria glabrata snail is, from a spatial point of view, detrimental to the host's digestive-genital gland complex growth. For mono- and plurimiracidial infections, the digestive gland volumes are, respectively 51 and 24% of those of control snails. Identical reduction of the infected genital gland volume (43% of controls) occurs in both cases. After the prepatent period, the ratio of parasite/digestive gland volumes (P/PDG) remains fairly constant at around 0.60 independent of the miracidial dose infection, indicative of a balanced host-parasite development which is discussed in relation to the spatial and energy constraints of this system.     

Interactions between the effects of atmospheric CO2 content and P nutrition on photosynthesis in white lupin (Lupinus albus L.)

Phosphorus (P) is a major factor limiting the response of carbon acquisition of plants and ecosystems to increasing atmospheric CO2 content. An important consideration, however, is the effect of P deficiency at the low atmospheric CO2 content common in recent geological history, because plants adapted to these conditions may also be limited in their ability to respond to further increases in CO2 content. To ascertain the effects of low P on various components of photosynthesis, white lupin (Lupinus albus L.) was grown hydroponically at 200, 400 and 750 micromol mol(-1) CO2, under sufficient and deficient P supply (250 and 0.69 microM P, respectively). Increasing growth CO2 content increased photosynthesis only under sufficient growth P. Ribulose 1,5-biphosphate carboxylase/oxygenase (Rubisco) content and activation state were not reduced to the same degree as the net CO2 assimilation rate (A), and the in vivo rate of electron transport was sufficient to support photosynthesis in all cases. The rate of triose phosphate use did not appear limiting either, because all the treatments continued to respond positively to a drop in oxygen levels. We conclude that, at ambient and elevated CO2 content, photosynthesis in low-P plants appears limited by the rate of ribulose biphosphate (RuBP) regeneration, probably through inhibition of the Calvin cycle. This failure of P-deficient plants to respond to rising CO2 content above 200 micromol mol(-1) indicates that P status already imposes a widespread restriction in plant responses to increases in CO2 content from the pre-industrial level to current values.     

Spatial and temporal variation in organic acid anion exudation and nutrient anion uptake in the rhizosphere of Lupinus albus L.

We investigated in situ the temporal patterns and spatial extent of organic acid anion exudation into the rhizosphere solution of Lupinus albus, and its relation with the nutrient anions phosphate, nitrate and sulfate by means of a rhizobox micro suction cup method under P sufficient conditions. We compared the soil solution in the rhizosphere of cluster roots with that in the vicinity of normal roots, nodules and bulk soil. Compared to the other rhizosphere and soil compartments, concentrations of organic acid anions were higher in the vicinity of cluster roots during the exudative burst (citrate, oxalate) and nodules (acetate, malate), while concentrations of inorganic nutrient anions were highest in the bulk soil. Both active cluster roots and nodules were most efficient in taking up nitrate and phosphate. The intensity of citrate exudation by cluster roots was highly variable. The overall temporal patterns during the lifetime of cluster roots were overlaid by a diurnal pattern, i.e. in most cases, the exudation burst consisted of one or more peaks occurring in the afternoon. Multiple exudation peaks occurred daily or were separated by 1 or 2 days. Although citrate concentrations decreased with distance from the cluster root apex, they were still significantly higher at a distance of 6 to 10 mm than in the bulk soil. Phosphate concentrations were extremely variable in the proximity of cluster roots. While our results indicate that under P sufficient conditions cluster roots take up phosphate during their entire life time, the influence of citrate exudation on phosphate mobilization from soil could not be assessed conclusively because of the complex interactions between P uptake, organic acid anion exudation and P mobilization. However, we observed indications of P mobilization concurrent with the highest measured citrate concentrations. In conclusion, this study provides semiquantitative in situ data on the reactivity of different root segments of L. albus L. in terms of root exudation and nutrient uptake under nutrient sufficient conditions, in particular on the temporal variability during the lifetime of cluster roots.     

Interaction between Orobanche crenata and its host legumes: unsuccessful haustorial penetration and necrosis of the developing parasite

BACKGROUND AND AIMS: Orobanche species represent major constraints to crop production in many parts of the world as they reduce yield and alter root/shoot allometry. Although much is known about the histology and effect of Orobanche spp. on susceptible hosts, less is known about the basis of host resistance to these parasites. In this work, histological aspects related to the resistance of some legumes to Orobanche crenata have been investigated in order to determine which types of resistance responses are involved in the unsuccessful penetration of O. crenata. METHODS: Samples of resistance reactions against O. crenata on different genotypes of resistant legumes were collected. The samples were fixed, sectioned and stained using different procedures. Sections were observed using a transmission light microscope and by epi-fluorescence. KEY RESULTS: Lignification of endodermal and pericycle host cells seems to prevent parasite intrusion into the root vascular cylinder at early infection stages. But in other cases, established tubercles became necrotic and died. Contrary to some previous studies, it was found that darkening at the infection site in these latter cases does not correspond to death of host tissues, but to the secretion of substances that fill the apoplast in the host-parasite interface and in much of the infected host tissues. The secretions block neighbouring host vessels. This may interfere with the nutrient flux between host and parasite, and may lead to necrosis and death of the developing parasite. CONCLUSIONS: The unsuccessful penetration of O. crenata seedlings into legume roots cannot be attributed to cell death in the host. It seems to be associated with lignification of host endodermis and pericycle cells at the penetration site. The accumulation of secretions at the infection site, may lead to the activation of xylem occlusion, another defence mechanism, which may cause further necrosis of established tubercles.     

Trypanosoma cruzi: effect of phenothiazines on the parasite and its interaction with host cells.

Phenothiazines were observed to have a direct effect on Trypanosoma cruzi and on its in vitro interaction with host cells. They caused lysis of trypomastigotes (50 uM/24 h) and, in axenic medium, dose-dependent inhibition of amastigote and, to a lesser extent, epimastigote proliferation. Treatment of infected peritoneal macrophages with 12.5 uM chlorpromazine or triflupromazine inhibited the infection; this effect was found to be partially reversible if the drugs were removed after 24 h of treatment. At 60 uM, the drugs caused damage to amastigotes interiorized in heart muscle cells. However, the narrow margin of toxicity between antitrypanosomal activity and damage to host cells mitigates against in vivo investigation at the present time. Possible hypotheses for the mechanism of action of phenothiazines are discussed.