Root Carbon Dioxide Fixation by Phosphorus-Deficient Lupinus albus (Contribution to Organic Acid Exudation by Proteoid Roots)

When white lupin (Lupinus albus L.) is subjected to P deficiency lateral root development is altered and densely clustered, tertiary lateral roots (proteoid roots) are initiated. These proteoid roots exude large amounts of citrate, which increases P solubilization. In the current study plants were grown with either 1 mM P (+P-treated) or without P (-P-treated). Shoots or roots of intact plants from both P treatments were labeled independently with 14CO2 to compare the relative contribution of C fixed in each with the C exuded from roots as citrate and other organic acids. About 25-fold more acid-stable 14C, primarily in citrate and malate, was recovered in exudates from the roots of -P-treated plants compared with +P-treated plants. The rate of in vivo C fixation in roots was about 4-fold higher in -P-treated plants than in +P-treated plants. Evidence from labeling intact shoots or roots indicates that synthesis of citrate exuded by -P-treated roots is directly related to nonphotosynthetic C fixation in roots. C fixed in roots of -P-treated plants contributed about 25 and 34% of the C exuded as citrate and malate, respectively. Nonphotosynthetic C fixation in white lupin roots is an integral component in the exudation of large amounts of citrate and malate, thus increasing the P available to the plant.     

Sucrose Metabolism in Lupinus albus L. Under Salt Stress

Salt stress (50 and 150 mM NaCl) effects on sucrose metabolism was determined in Lupinus albus L. Sucrose synthase (SS) activity increased under salt stress and sucrose phosphate synthase activity decreased. Acid invertase activity was higher at 50 mM NaCl and decreased to control levels at 150 mM NaCl. Alkaline invertase activity increased with the salt stress. Glucose content decreased with salt stress, sucrose content was almost three times higher in plants treated with 150 mM NaCl and fructose content did not change significantly. The most significant response of lupin plants to NaCl excess is the increase of sucrose content in leaves, which is partially due to SS activity increase under salinity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytokinin activity in Lupinus albus

The distribution and metabolism of {8-¹⁴C}zeatin incorporated into the transpiration stream of fruiting white lupin plants ( Lupinus albus L.) has been studied. The distribution pattern of ¹⁴C in the different aerial organs suggests that the amount of cytokinin being incorporated into any one organ may have been a function of its transpiration rate. Once in these organs, particularly the leaves, zeatin was rapidly metabolised and or utilised. This resulted in the formation of a number of labelled compounds that did not give a response with the soybean callus bioassay. Substances co-eluting with zeatin glucoside and ribosylzeatin appeared to be the principal biologically active metabolites. From the present evidence it can be concluded that the leaf and side shoots received a major proportion of the applied labelled cytokinin. However, the presence of a small amount of radioactivity co-eluting with zeatin and ribosylzeatin in the fruits indicates that the high levels of cytokinins normally associated with these organs need not necessarily all have been synthesised in situ.     

The Response of Lupinus albus Roots to the Signal from Phosphorus-Deficient Substrate1

The excretion of organic acids into the rhizosphere is induced by low phosphorus content in roots of white lupine (Lupinus albus L.). The aim of this study was to investigate how did the white lupine roots respond to the signals of P-deficiency in the substrate, by using the method of separating the root system into two parts, one part being placed into P-deficient solution and another part into P-sufficient solution. The results showed that the root tips (1-cm-long) accept the signal of P-deficiency in the substrate. This signal can be transmitted from one root tip to another, and can induce the accumulation and excretion of malic acid in the root tips and the development of proteoid roots. In order to investigate whether a phytohormone was involved in the response, the external hormones were used. Indole-3-butyric acid added to P-sufficient solution can induce the development of proteoid roots, but without any accumulation of citric acid in them. On the contrary, 6-benzyladenine (BA) added to the P-deficient solution can inhibit the development of proteoid roots and the accumulation of malic acid in the root tips. The inhibitory effect increases with increasing BA supply.     

Cytokinin Activity in Lupinus albus

The cytokinin content of the root exudate and leaves of fruiting white lupin plants (Lupinus albus L.) was investigated at 2 weekly intervals after anthesis of the lowest flower on the primary inflorescence. Up to 8 weeks after anthesis the level of cytokinins in the root exudate increased. However, at 10 weeks after anthesis insufficient sap was produced for analysis. Cytokinins co-eluting with zeatin and zeatin riboside were detected in the root exudate after fractionation on Sephadex LH-20. The cytokinin levels in the mature leaves steadily increased up to 8 weeks after anthesis and thereafter remained relatively constant. Three peaks of activity, co-eluting with zeatin, zeatin riboside and the glucoside cytokinins were recorded in the leaf extracts. The level of glucoside cytokinins in the leaves was high at 8 and 10 weeks after anthesis. Paper chromatography of extracts of fruits collected at 2 weeks after anthesis indicated that as fruit development proceeded there was a build up of cytokinin in this region of the plant. It is suggested that, in the white lupin, the cytokinins translocated to the shoot are accumulated in the leaves and in the fruits and that it is only later when there is a considerable decrease in sap (10 weeks after anthesis) production that a decreasing supply of cytokinins leads to shoot senescence.     

Soil Phosphorus Uptake by Continuously Cropped Lupinus albus: A New Microcosm Design

When grown in soils with sparingly available phosphorus (P), white lupin (Lupinus albus L.) forms special root structures, called cluster roots, which secrete large amounts of organic acids and concomitantly acidify the rhizosphere. Many studies dealing with the understanding of this P acquisition strategy have been performed in short time experiments either in hydroponic cultures or in small microcosm designs with sand or sand:soil mixtures. In the present study, we applied an experimental design which came nearer to the natural field conditions: we performed a one-year experiment on large microcosms containing 7 kg of soil and allowing separation of rhizosphere soil and bulk soil. We planted six successive generations of lupins and analysed P uptake, organic P desorption, phosphatase activities and organic acid concentrations in different soil samples along a spatio-temporal gradient. We compared the rhizosphere soil samples of cluster (RSC) and non-cluster roots (RSNC) as well as the bulk soil (BS) samples. A total shoot biomass of 55.69 ± 1.51 g (d.w.) y⁻¹ was produced and P uptake reached 220.59 ± 5.99 mg y⁻¹. More P was desorbed from RSC than from RSNC or BS (P < 0.05). RSC and RSNC showed a higher activity of acid and alkaline phosphatases than BS samples and a higher acid phosphatase activity was observed in RSC than in RSNC throughout the one-year experiment. Fumarate was the most abundant organic acid in all rhizosphere soil samples. Citrate was only present in detectable amounts in RSC while malate and fumarate were recovered from both RSC and RSNC. Almost no organic acids could be detected in the BS samples. Our results demonstrated that over a one-year cultivation period in the absence of an external P supply, white lupin was able to acquire phosphate from the soil and that the processes leading to this P uptake took place preferentially in the rhizosphere of cluster roots.     

Phosphorus deficiency affects the allocation of below-ground resources to combined cluster roots and nodules in Lupinus albus

Lupins can rely on both cluster roots and nodules for P acquisition and biological nitrogen fixation (BNF), respectively. The resource allocation (C, N and P) between cluster roots and nodules has been largely understudied during P-deficient conditions. The aim of this investigation was therefore to determine the changes in resource allocation between these organs during fluctuations in P supply. Lupinus albus was cultivated in sand culture for 3 weeks, with either sufficient (2 mM high) or limiting (0.1 mM low) P supply. Although variation on P supply had no effect on the total biomass, there were significant differences in specialised below-ground organ allocation to cluster roots and nodule formation. Cluster root formation and the associated C-costs increased during low P supply, but at sufficient P-supply the construction and growth respiration costs of cluster roots declined along with their growth. In contrast to the cluster root decline at high P supply, there was an increase in nodule growth allocation and corresponding C-costs. However, this was not associated with an increase in BNF. Since cluster roots were able to increase P acquisition under low P conditions, this below-ground investment may also have benefited the P nutrition of nodules. These findings provide evidence that when lupins acquire N via BNF in their nodules, there may be a trade-off in resource allocation between cluster roots and nodules.     

缺磷白羽扇豆排根与非排根区根尖分泌有机酸的比较

采用根系分泌有机酸原位收集方法及高效液相色谱技术分析了供磷及缺磷后不同时间白羽扇豆(LupinusalbusL .)非排根区根尖和排根分泌有机酸的种类和数量 ,以及相应的根尖、排根组织 ,茎木质部、韧皮部汁液中有机酸含量的变化。结果表明 :(1)缺磷能够诱导白羽扇豆根系产生大量排根 ,根系的有机酸分泌量也明显增加。 (2 )无论在供磷或缺磷条件下 ,排根与非排根区根尖组织中的有机酸种类相同 ,但排根主要分泌柠檬酸和苹果酸 ,而非排根区根尖主要分泌苹果酸和乙酸。 (3)缺磷后非排根区根尖分泌苹果酸的量增加 ,至第 17天达到高峰 ;排根开始分泌柠檬酸的时间相对较晚。缺磷后排根分泌柠檬酸的量随缺磷时间的延长不断增加。 (4 )在缺磷的排根与非排根区根尖组织和茎木质部伤流液中含有大量柠檬酸和苹果酸 ,但在茎韧皮部汁液中则几乎检测不到这两种有机酸。上述结果表明 ,尽管排根和非排根区根尖组织中的有机酸种类相同 ,但它们向外分泌的有机酸种类不同。缺磷后排根及非排根区根尖增加向外分泌的有机酸主要在根中合成     

Ein eigenartiger Wachstumsmodus der Hauptwurzeln bei Lupinus albus

Ohne Zusammenfassung     

Characterization of isoperoxidase-B2 inactivation in etiolated Lupinus albus hypocotyls

One basic peroxidase isoenzyme, with a pI of 8.8, is present in the intercellular washing fluid in the aerial part of 6-day-old Lupinus albus hypocotyl seedlings. This isoenzyme, called LuP-B2, is the principal soluble component secreted into the apoplastic space and it is a constitutive enzyme along the whole length of etiolated hypocotyl. The enzymatic inactivation process which this apoplastic peroxidase undergoes is described for the first time. The kinetic constants which describe its inactivation by H(2)O(2) in the absence of reductant substrates are determined. LuP-B2 is inactivated in situ and in vitro in a time- and concentration-dependent manner. H(2)O(2) acts as a suicide substrate according to a model previously proposed by us. The constant values calculated are similar to those calculated for the basic isoenzyme of horseradish roots, HRP-C. LuP-B2 presents a k(inact) value of 7.5 x 10(-3) s(-1) and a k(cat) of 6.7 s(-1). This isoenzyme makes 889 catalytic cycles for each inactivation event. The similarity in behavior and the constant values, together with other situations (both are excreted, soluble and constitutive isoenzymes) suggest that the inactivation process could play an important role in plant development and stress situations.     

A Cytokinin Complex in the Developing Fruits of Lupinus albus

The apices of Lupinus albus L. were analysed for cytokinin activity at three stages of development. Little cytokinin activity could be detected in the apices at the time of flowering. However, a considerable amount of activity was detected as the fruits developed. Separate analyses of seed and pod material indicated that there was a high level of cytokinin in both these parts of the fruit. After fractionation of the peaks of activity obtained from paper chromatograms on Sephadex LH-20, four peaks of cytokinin activity were recorded. Two of these co-eluted with zeatin and zeatin riboside. A third peak at an elution volume of 360–440 ml could be hydrolysed with β-glucosidase to give activity at elution volumes corresponding to those of zeatin and zeatin riboside. This strongly suggested that both glucosylated zeatin and glucosylated zeatin riboside were present in the developing fruits of L. albus. The fourth peak at an elution volume of 160–280 ml did not disappear upon hydrolysis with β-glucosidase, and it is possible that it represented a nucleotide cytokinin.     

Changes in lipoxygenase activity during seedling development of Lupinus albus

A lipoxygenase preparation was obtained from Lupinus albus seeds and was shown to differ from previously characterized lipoxygenase. This study describes changes in lipoxygenase activity during seedling development of Lupinus albus. The enzyme activity shows a decrease from 0–6 h postgermination (about 15%), is roughly constant or even rises slightly from 6–30 h and then shows a large increase between 30 and 48 h (about 50%). Enzymatically active proteins from 48 h-old seedlings were isolated and the increase of enzyme activity was mainly due to the presence of two components with maximum activity at pH 6 and pH 8.5, respectively. When arachidonic acid was used as substrate, the two enzymatic activities produce 15 HPETE. The increase in lipoxygenase activity during seedling development was inhibited by cycloheximide. Cordycepin appears to have no direct effect on lipoxygenase synthesis in vivo at the studied doses.     

Leghemoglobin in Lupin Plants (Lupinus albus cv Multolupa)

Leghemoglobin was localized by immunogold techniques in nodules of Lupinus albus cv Multolupa inoculated with Bradyrhizobium sp. (Lupinus) strain ISLU 16. The protein localization was performed in nodules embedded in Spurr's and Araldite epoxy resins and Lowycryl K4M. A very good preservation of both the ultrastructure and antigenicity was obtained with the tissues embedded in Araldite following glutaraldehyde fixation and unpostfixed in osmium tetroxide. Lupin leghemoglobin is a stable and abundant protein which allows a conventional method to be safely used for localization of leghemoglobin. Labeling of leghemoglobin was specifically confined to the cytosol matrix and nuclei. Gold particles were never observed in the peribacteroidal spaces nor in the cytoplasmic organelles of the infected cells. Decrease of leghemoglobin was observed when the plants were grown with 10.7 micromolar and 21.4 micromolar of nitrate.     

Root Cluster Formation and Citrate Exudation of White Lupin （Lupinus albus L.） as Related to Phosphorus Availability

A split-root system was used to investigate whether the external or internal P concentration controls root cluster formation and citrate exudation in white lupin (Lupinus albus L.) grown under controlled conditions. In spite of low P concentrations in the shoots and roots of the -P plant, its dry weight was not reduced compared with the P plant. Supplying external P (0.25 mmol/L) to one root half resulted in an increase in P concentration not only in the shoot, but also in the P-deprived root half, indicating P cycling within the plants. Omitting P from both split-root pots stimulated root cluster formation in both root halves,whereas P supply to one root half stimulated root cluster formation at the beginning of the treatment. Neither P supply to just one root half continuously nor resupply of P to one root half after 19 d of P starvation inhibited root cluster formation on the P-deprived side, although the concentration of P in this root half and shoot increased markedly. The results indicate that root cluster formation in L. albus is controlled by both shoot and root P concentrations. The rates of citrate exudation by both root halves with P deficiency were higher than those of the one root half supplied with P only. In the treatment with one root half supplied with P, the rates of citrate exudation by either the P-supplied or -deprived root halves were almost the same,regardless of P concentration in the roots. The results suggest that internal P concentration controls root cluster formation and citrate exudation in white lupin, but these processes may be regulated by different mechanisms.     

Root Cluster Formation and Citrate Exudation of White Lupin (Lupinus albus L.) as Related to Phosphorus Availability

A split-root system was used to investigate whether the external or internal P concentration controls root cluster formation and citrate exudation in white lupin (Lupinus albus L.) grown under controlled conditions. In spite of low P concentrations in the shoots and roots of the -P plant, its dry weight was not reduced compared with the P plant. Supplying external P (0.25 mmol/L) to one root halfresulted in an increase in P concentration not only in the shoot, but also in the P-deprived root half, indicating P cycling within the plants. Omitting P from both split-root pots stimulated root cluster formation in both root halves,whereas P supply to one root halfstimulated root cluster formation at the beginning of the treatment. Neither P supply to just one root half continuously nor resupply of P to one root half after 19 d of P starvation inhibited root cluster formation on the P-deprived side, although the concentration of P in this root half and shoot increased markedly. The results indicate that root cluster formation in L. albus is controlled by both shoot and root P concentrations. The rates of citrate exudation by both root halves with P deficiency were higher than those of the one root half supplied with P only. In the treatment with one root half supplied with P, the rates of citrate exudation by either the P-supplied or -deprived root halves were almost the same,regardless of P concentration in the roots. The results suggest that internal P concentration controls root cluster formation and citrate exudation in white lupin, but these processes may be regulated by different mechanisms.     

Fungitoxic dihydrofuranoisoflavones and related compounds in white lupin,Lupinus albus

Chromatographic investigation of a methanolic extract of white lupin roots has revealed the presence of six new dihydrofuranoisoflavones (lupinisoflavones A-F). Three monoprenylated (3,3-dimethylallyl-substituted) isoflavones (wighteone, luteone and licoisoflavone A), two diprenylated isoflavones [6,3′-di(3,3-dimethylallyl)genistein (lupalbigenin) and 6,3′-di(3,3-dimethylallyl)-2′-hydroxygenistein (2′-hydroxylupalbigenin)] and two pyranoisoflavones (parvisoflavone B and licoisoflavone B) have also been isolated from the same source. In addition to genistein, leaf extracts of L. italbus contain 3′-O-methylorobol which is presumed to be the precursor of lupisoflavone [5,7,4′-trihydroxy-3′-methoxy-6-(3,3-dimethylallyl)isoflavone]. Probable biogenetic relationships between the prenylated, and dihydrofurano-and pyrano-substituted isoflavones in roots and leaves of L. albus are briefly discussed.