Modelling of the flows and partitioning of carbon and nitrogen in the holoparasite Cuscuta reflexa Roxb. and its host Lupinus albus L.: II. Flows between host and parasite and within the parasitized host

A recently developed empirically based modelling technique wasused to quantify uptake, flow and utilization of C and N inLupinus albus L., uninfected and parasitized by Cuscuta reflexaRoxb. plants over a 12 d period during flowering and early fruitsetting of the host. The modelling combined data on molar C:Nratios in host phloem and pressure-induced xylem sap, net incrementsof C and N in host and parasite plant parts and respiratorylosses of C. The modelling of the solute transfer between hostand Cuscuta was achieved by assuming non-specific intake fromthe xylem. The models predicted that Cuscuta derived 99.5% ofits carbon and 93.6% of its nitrogen demand from the host phloem.The overriding sink strength of the parasite diverted most ofthe basipetally translocated host assimilates and massivelycompeted with the host root and inhibited fruit setting. Carbonincorporation in Cuscuta consumed 56%, respiration 24% and secretionby extrafloral nectaries 1.8% of the current host photosynthate.Root respiration was inhibited by 59% and carbon was mobilizedfrom host root and leaves. Competition by the parasite for Nwas even more severe and Cuscuta incorporated nitrogen equalling223% of current fixation, but N₂ fixation of the host was severelyrestricted to 37%. Withdrawal of N from host phloem led to severelosses of N from leaves and the root and marked decreases inN concentration. It required massive xylem-to-phloem transferof N, because the xylem as the major supply route for N wasnot exploited substantially by Cuscuta. The results are discussedin relation to likely causes for parasite-induced pathogeniceffects, suggesting that Cuscuta affected the host adverselyby depriving it mainly of its nitrogen, but that causal to incipientnitrogen deficiency and restricted N2 fixation was the superiorsink potential of Cuscuta, which prevented adequate supply ofassimilates to the nodulated root. The dominating sink potentialof Cuscuta is compared with the similarly strong sink competitionexerted by fruits at the stage of seed filling in annual plants. Key words: Cuscuta reflexa, Lupinus albus, parasitism, carbon, nitrogen, phloem, xylem, transport     

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.     

Soil-plant water relations, root distribution and biomass partitioning in Lupinus albus L. under drought conditions

Lupinus albus L. from different climatic origins responded toa 15 d period of water shortage during flowering by losing 50%of the total leaf canopy and gaining 55% in stem dry weight.Water deficits also led to a significant increase in the fineroot length density and a slight increase in the fine root dryweight. The latter increase was especially pronounced in thedeeper soil layers. Some marginal differences among genotypeswere observed in the responses. Stomatal closure by midday wasan early response to water deficit, giving rise to constantpredawn leaf water potentials during the first week of watershortage in spite of a decrease of 60% in the available soilwater. No osmotic regulation or adjustments of the cell wallproperties were observed in any of the lupin lines. We explainedthe maintenance of seed production in water-stressed plantsby their ability to accumulate assimilates in the shoot, whichwould be diverted to the pods during the seed filling stage. Key words: Biomass partitioning, drought, Lupinus albus, root distribution, water relations.     

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.     

Population structure and linkage disequilibrium in Lupinus albus L. germplasm and its implication for association mapping

White lupin (Lupinus albus L.) has been around since 300 B.C. and is recognized for its ability to grow on poor soils and application as green manure in addition to seed harvest. The seed has very high levels of protein (33-47 %) and oil (6-13 %). It also has many secondary metabolites that are potentially of nutraceutical value to animals and humans. Despite such a great potential, lupins role in modern agriculture began only in the twentieth century. Although a large collection of Lupinus germplasm accessions is available worldwide, rarely have they been genetically characterized. Additionally, scarce genomic resources in terms of recombinant populations and genome information have been generated for L. albus. With the advancement in association mapping methods, the natural populations have the potential to replace the recombinant populations in gene mapping and marker-trait associations. Therefore, we studied the genetic similarity, population structure and marker-trait association in a USDA germplasm collection for their current and future application in this crop improvement. A total of 122 PI (Plant Inventory) lines were screened with 18 AFLP primer pairs that generated 2,277 fragments. A subset of 892 polymorphic markers with MAF >0.05 (minor allele frequency) were used for association mapping. The cluster analysis failed to group accessions on the basis of their passport information, and a weak structure and low linkage disequilibrium (LD) were observed indicating the usefulness of the collection for association mapping. Moreover, we were also able to identify two markers (a p value of 1.53 × 10(-4) and 2.3 × 10(-4)) that explained 22.69 and 20.5 % of seed weight variation determined using R (LR) (2) . The implications of lack of geographic clustering, population structure, low LD and the ability of AFLP to map seed weight trait using association mapping and the usefulness of the PI collections in breeding programs are discussed.     

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.     

Hidden in the host – Unexpected vegetative hibernation of the holoparasite Cuscuta epithymum (L.) L. and its implications for population persistence

Several reports have referred to the possible perennial character of holoparasitic Cuscuta species, but the frequency and ecological importance of perennation have not been studied yet. We determined that Cuscuta epithymum is capable of overwintering vegetatively, especially on its most common perennial host Calluna vulgaris. To examine the impact of successional stages on the capability of C. epithymum to perennate vegetatively, ten C. epithymum populations in Calluna-dominated sites in a successional gradient were studied. Although the number of overwintering haustoria varied between different populations, on average 85% of each C. epithymum population was the result of resprouting haustoria. Thus, the pseudoannual growth habit is an important, but overlooked life-strategy for long-term survival of this species. Furthermore, the stage of heath succession significantly determined the overwintering probabilities of C. epithymum and therefore strongly influenced its annual growth. Most sprouted haustoria were found in 1- or 2-year-old vegetation followed by a gradual decline at older successional stages. The parasite was more likely to overwinter on unlignified (and nutritious) than on lignified parts of C. vulgaris. The number of sprouted haustoria positively affects C. epithymum population size and thus flower abundance. Hence, vegetative perennation and subsequent increased flowering success are two aspects that may increase the chance for these populations to cope with environmental influences and survive in the long run.     

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 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.     

Turnover and transport of quinolizidine alkaloids. Diurnal fluctuations of lupanine in the phloem sap,leaves and fruits of Lupinus albus L.

Quinolizidine alkaloids formed in the leaves of Lupinus albus L. are translocated via the phloem to the other plant organs, especially the maturing fruits. Compared with amino-acid transport in the phloem, the alkaloids contribute about 8% to the overall nitrogen being exported from the leaf. Since it is likely that the alkaloids are subsequently degraded in the target tissues a minor role of quinolizidine alkaloids might be nitrogen transport. A marked diurnal fluctuation of alkaloids was observed in the leaves, the phloem sap, the roots and the fruits with an increase during the day and an amplitude of several hundred percent thus providing evidence for a rapid turnover of endogenous alkaloids.Abbreviations QA quinolizidine alkaloids - GLC gas-liquid chromatography     

Acclimation of photosynthesis to low temperature in Spinacia oleracea L. I. Effects of acclimation on CO2 assimilation and carbon partitioning

Acclimation of spinach plants grown at 25C to a temperatureof 10C for 10 d resulted in an increased capacity for leafphotosynthesis in saturating light and CO₂ but not at ambientCO₂ concentrations. Gas exchange and chlorophyll fluorescencemeasurements indicated that acclimation was accompanied by anincreased capacity for the regeneration of ribulose-1,5-bisphosphate.Changes in starch, soluble carbohydrates and activities of sucrose-Psynthase and ADP-glucose pyrophosphorylase were measured duringthe acclimation process. There was an initial increase in starchand sucrose during the first 2 d, but these then declined. Therewas an increase in the capacity for sucrose synthesis duringlow temperature acclimation, evidenced by an increase in themaximum activity of sucrose-P synthase activity and an increasein partitioning of ¹⁴CO₂ into sucrose, but there was no increasein the activity of ADP-glucose pyrophosphorylase or carbon partitioninginto starch. Key words: Acclimation, carbon metabolism, gas exchange, low temperature, spinach, Spinacia oleracea     

Sink-stimulated photosynthesis and sink-dependent increase in nitrate uptake: nitrogen and carbon relations of the parasitic association Cuscuta reflexa–Ricinus communis

Ricinus communis L. was grown under limiting N supply in quartz sand culture, fed with 0.2, 1 or 5 mol m^?3 NO₃^?, or in liquid culture with 0.022, 0.05 or 0.5 mol m^?3 NO₃^?. Some of the plants were infected with Cuscuta reflexa Roxb. As occurred for the host, dry matter production and growth of C. reflexa were severely depressed with decreasing N supply to the host. When parasitized by C. reflexa, the shoot and root dry weight of Ricinus was diminished at all levels of N nutrition, but the total dry weight of host plus parasite was almost the same as that of uninfected Ricinus. In contrast to the situation in Lupinus albus (Jeschke et al. 1994b), infection by Cuscuta resulted in increased tissue N levels in the host and the N content of the system Ricinus plus C. reflexa was the same or even somewhat larger than that of uninfected plants. This indicated a sink-dependent stimulation of nitrate uptake. As a result of decreased root weights, nitrate uptake g^?1 FW was stimulated by 80, 60 or only 40% at 0.2, 1 or 5 mol m^?3 nitrate supply. Increased nitrate uptake was reflected, particularly at low N supply, in xylem transport; xylem sap nitrate concentrations were substantially elevated, while those of amino acids were decreased in parasitized plants. This indicated an inhibition of nitrate assimilation in roots of parasitized plants under limiting N supply. Besides these effects on N relations, C. reflexa induced a substantial sink-dependent stimulation of net photosynthesis in host leaves and a concomitant increase in stomatal opening and transpiration. This stimulation depended on the relative sink size induced by Cuscuta, on nitrogen nutrition and on leaf age, indicating that delayed senescence of leaves contributes to the overall effects of Cuscuta on its host. The Cuscuta-induced inhibition of nitrate assimilation in the roots and the increase in nitrate uptake suggest that nitrate reduction was shifted towards the leaves in the presence of C. reflexa. The stimulating effects of C. reflexa in the Ricinus-Cuscuta association are compared with the strongly inhibitory effects occurring in the tripartite association L. albus–Rhizobium–Cuscuta reflexa.     

Comparison of the response to phosphorus deficiency in two lupin species,Lupinus albus and L. angustifolius,with contrasting root morphology

White lupin (Lupinus albus) produces cluster roots, an adaptation to low soil phosphorus (P). Cluster roots exude large levels of P‐solubilizing compounds such as citrate and malate. In contrast, narrow leaf lupin (L. angustifolius) is closely related to L. albus, but does not produce cluster roots. To examine the different strategies for P acquisition, we compared the growth, biomass allocation, respiratory properties and construction cost between L. albus and L. angustifolius under P‐deficient conditions. Both Lupinus species were grown in hydroponic culture with 1 or 100 μM P. Under the P‐deficient regime, L. albus produced cluster roots with little change in biomass allocation, while L. angustifolius significantly increased biomass allocation to roots. The rate of cyanide‐resistant SHAM (salicylhydroxamic acid)‐sensitive respiration was high in cluster roots and very low in roots of L. angustifolius. These results suggest a low alternative oxidase (AOX) activity in L. angustifolius roots, and thus, ATP would be produced efficiently in L. angustifolius roots. The construction cost was highest in cluster roots and lowest in L. angustifolius roots. This study shows that under P deficiency, L. albus produces high‐cost cluster roots to increase the P availability, while L. angustifolius produces large quantities of low‐cost roots to enhance P uptake.     

The first genetic and comparative map of white lupin (Lupinus albus L.): identification of QTLs for anthracnose resistance and flowering time, and a locus for alkaloid content.

We report the first genetic linkage map of white lupin (Lupinus albus L.). An F8 recombinant inbred line population developed from Kiev mutant x P27174 was mapped with 220 amplified fragment length polymorphism and 105 gene-based markers. The genetic map consists of 28 main linkage groups (LGs) that varied in length from 22.7 cM to 246.5 cM and spanned a total length of 2951 cM. There were seven additional pairs and 15 unlinked markers, and 12.8% of markers showed segregation distortion at P < 0.05. Syntenic relationships between Medicago truncatula and L. albus were complex. Forty-five orthologous markers that mapped between M. truncatula and L. albus identified 17 small syntenic blocks, and each M. truncatula chromosome aligned to between one and six syntenic blocks in L. albus. Genetic mapping of three important traits: anthracnose resistance, flowering time, and alkaloid content allowed loci governing these traits to be defined. Two quantitative trait loci (QTLs) with significant effects were identified for anthracnose resistance on LG4 and LG17, and two QTLs were detected for flowering time on the top of LG1 and LG3. Alkaloid content was mapped as a Mendelian trait to LG11.