JXB Advance Access originally published online on December 22, 2006
Journal of Experimental Botany 2007 58(5):935-946; doi:10.1093/jxb/erl254
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RESEARCH PAPER |
Synthesis, transport and accumulation of quinolizidine alkaloids in Lupinus albus L. and L. angustifolius L.
1School of Plant Biology, the University of Western Australia, Nedlands, Western Australia 6907, Australia
2Chemistry Centre of Western Australia, 150 Hay Street, East Perth, Western Australia 6007, Australia
* To whom correspondence should be addressed. E-mail: m.lee{at}murdoch.edu.au or catkins{at}cyllene.uwa.edu.au
Received 12 July 2006; Revised 24 October 2006 Accepted 30 October 2006
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Abstract |
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Each of the principal quinolizidine alkaloids (QA) found in both xylem and phloem exudates together with extracts from all component organs collected from bitter (cv. Lupini) and sweet (cv. Ultra) cultivars of Lupinus albus L. were quantified by gas chromatographic analyses throughout reproductive development. In addition to establishing the major translocated QA species estimates for fluxes of QA to developing fruits based on their sap composition and water economy showed that around half of the QA that accumulated in fruit tissues was due to synthesis in situ and half to translocation principally by phloem. Detailed analyses of QA in transport fluids and component organs were extended to reciprocal homo- and hetero-grafts using bitter (cv. Fest) and sweet (cv. Danja) cultivars of L. angustifolius L. These data confirmed that the majority of QA were synthesized in shoot tissues. In both lupin species feeding and analysis of deuterated QA (lupanine and 13-hydroxylupanine) were used as tracers to demonstrate direct redistribution of alkaloids by translocation from mature leaves in phloem.
Key words: Alkaloid, GC/MS, Lupinus, quinolizidine, synthesis, transport
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Quinolizidine alkaloids (QA) are prominent among species of the ‘genistoid alliance’ group of legumes (tribes Genisteae, Crotalarieae, Podalyrieae, Liparieae, Thermopsideae, Euchresteae, Brongniartieae, and Sophoreae) (Wink, 2003) and are commonly referred to as ‘lupin alkaloids’, since they occur throughout species of the genus Lupinus (Mears and Mabry, 1971). Their distribution in legumes is of systematic significance at a relatively high taxonomic level (Crisp et al., 2000) and, together with phylogenetic analysis based on DNA sequence, have been used to re-examine membership of the genistoids (Käss and Wink, 1994, 1997; Wink and Waterman, 1999; Kite and Pennington, 2003; Wink and Mohamed, 2003). QA have antiviral, antibacterial, and antifungal properties (Wink, 1984, 1987b; Wippich and Wink, 1985) and are toxic to insects (Wink, 1986; Wink and Mende, 1987). The bitter taste imparted by the alkaloids acts as a deterrent to larger mammalian grazers, which might not initially be affected by pharmacological side-effects of eating lupins (Petterson et al., 1991), and ‘bitterness’ also reduces their palatability as animal feed (Edwards and van Barneveld, 1998).
Cultivated lupins, including L. albus, L. luteus, L. mutabilis, and L. angustifolius, have been selected and bred to produce ‘sweet’ varieties which have much lower concentrations of QA and, consequently, produce seed that is less toxic and more palatable. However, these varieties contain low levels of QA in vegetation as well as seed and not surprisingly have considerably lower resistance to disease and predation compared with ‘bitter’, wild germplasm (Wink, 1990a). The lowered alkaloid content of vegetative tissues has increased susceptibility to aphid attack and transmission of aphid-borne viruses (Sweetingham et al., 1998). To counter this inadequacy, Gladstones (1994) used a breeding strategy in L. angustifolius which selected for best possible resistance to aphids, (and thus QA content in the green vegetation among other anti-herbivore properties) while simultaneously selecting for least possible QA content in harvested seed. While this has not led to cultivars with sufficiently bitter foliage to deter aphids, but with seeds low in QA, such a ‘bitter/sweet’ phenotype is a feature of rough-seeded lupin species and particularly L. cosentinii (Gladstones, 1974). Whether there is lower QA synthesis in seeds or reduced translocation of QA to seeds in rough-seeded species, these observations raise the possibility that mutation or direct genetic manipulation could generate a similar phenotype in sweet cultivars of crop species. There have been no data for the degree to which lupin species, whether bitter or sweet, form QA in situ in seeds and the relative degree to which seeds rely on translocation from sites of synthesis elsewhere in the plant.
Despite numerous studies to establish the site(s) of QA biosynthesis in a range of lupin species there is still no clear resolution. Both grafting studies (Peters et al., 1957; Waller and Nowacki, 1978) and labelling experiments (Schütte, 1969; Wink, 1987b) have concluded that synthesis occurs predominantly in the aerial, green parts of the plant, and enzymic analysis provided evidence of a specific role for chloroplasts in the synthesis of the precursor lysine and in subsequent reactions specific to the primary QA pathway (Mazelis et al., 1976; Mills and Wilson, 1978; Wink and Hartmann, 1981). While the first enzyme specific to the QA synthesis, lysine decarboxylase, had higher activity in roots compared to leaflets of L. polyphyllus (Wink and Hartmann, 1981), subsequent enzymes of the pathway present in leaflets were absent in root extracts. Recently, Babaoglu et al. (2004) transformed roots of L. mutabilis with Agrobacterium rhizogenes and found that the ‘hairy’ roots synthesized enhanced levels of isoflavones but not QA. The authors confirmed earlier studies using root tissues (Wink, 1987c) and the observations provided further evidence that roots of lupin do not synthesize this group of alkaloids.
Phloem translocation is generally believed to be responsible for the subsequent distribution of QA from leaves to other sites where they finally accumulate, particularly in reproductive organs, and there have been a number of reports that confirm the presence of QA in phloem exudates from lupins (Wink and Witte, 1984; Bäumel and Jeschke, 1993; Jeschke et al., 1994; Bäumel et al., 1995). QA have also been measured in xylem fluids collected by pressure displacement from L. angustifolius (Bäumel et al., 1993) and L. albus (Bäumel et al., 1995). While the concentrations were much lower in xylem compared with phloem (0.15 versus 4.8 mM), the high rate of water movement in the transpiration stream compared to that in phloem led Bäumel et al. (1995) to conclude that xylem was a major route for QA distribution in white lupin. These authors extended their findings by using an empirical modelling approach (based on Pate et al., 1979) to estimate net flows of QA in xylem and phloem to component organs. The outcomes predicted the root to be the main site of biosynthesis (Bäumel et al., 1995). While earlier grafting studies (Moshkov and Smirnova, 1940) are consistent with this conclusion, more recent grafting experiments (Peters et al., 1957; Waller and Nowacki, 1978) are consistent with leaves being the major site for QA synthesis.
The present study provides detailed analyses for the range of QA found in both xylem and phloem exudates together with component tissues collected from bitter and sweet cultivars of L. albus during the period of reproductive development. Similar data for the cytokinins found in white lupin were used recently to estimate in situ synthesis and translocated flux for this group of plant growth regulators to seeds (Emery et al., 2000), and this approach is applied to QA. Because of a lack of agreement in the current literature about the significance of xylem versus phloem translocation of QA, and the relatively new hypothesis, that roots are a major site for QA synthesize in lupins (Bäumel et al., 1995), detailed analyses of transport fluids and component organs were extended to reciprocal homo- and hetero-grafts using bitter and sweet cultivars of L. angustifolius. Deuterated QAs were synthesized and used as tracers to more clearly and definitively describe processes of alkaloid redistribution by translocation in both lupin species.
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Materials and methods |
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Plant material
L. angustifolius L. (cv. Fest and cv. Danja) and L. albus L. (cv. Lupini and cv. Ultra) seed were obtained from the Department of Botany at the University of Western Australia, Perth. Plants were inoculated with a peat-based suspension of Bradyrhizobium spp. strain WU 425, grown in sand culture in a naturally-lit greenhouse from May to October in Perth, Western Australia, and supplied throughout growth with a nutrient solution free of combined N (Hocking and Pate, 1978).
Phloem exudate sampling
Prior to harvest, phloem exudate was collected from fruit tips, upper and lower stem and from leaf petioles using methods described earlier (Pate et al., 1974). Where fruit tip phloem exudate was collected, the stylar tip of the developing fruit was removed, and the first droplet of exudate was discarded, subsequent exudate was collected with a glass capillary, typically 500 µl was collected. Where stem and petiole phloem exudate was sampled, a shallow incision was made and the phloem exudate was collected with a glass capillary, typically 200 µl was collected. All phloem exudate samples were immediately frozen (–20 °C) after collection.
Plant harvest
Following phloem exudate sampling plants were decapitated approximately 2 cm above the level of the soil. Eight plants of L. albus (cv. Ultra and cv. Lupini) were harvested every 7 d from 73–115 DAS (cv. Ultra) or 94–143 DAS (cv. Lupini). In each case, these periods coincided with reproductive development starting with anthesis on the primary inflorescence and ending at seed maturity. Aerial parts of the harvested plants were divided into an upper and a lower half and the component organs of each separated into leaves, stems, petioles, and fruits. Fruits were further separated into pods and seeds where possible. The separate parts from all eight plants were pooled, rinsed with deionized water, immediately frozen (–20 °C) and freeze-dried.
Rootstock xylem exudate sampling
Xylem exudate was collected from the rootstock as described previously (Bollard, 1960). After the initial drop of xylem exudate was discarded, xylem exudate bleeding from the rootstock was collected for approximately 30 min with a glass capillary tube, typically 1 ml of exudate was collected which was then immediately frozen (–20 °C).
Grafting experiments
Seedling cleft grafts were established involving 21-d-old shoots of the sweet (cv. Danja) or bitter (cv. Fest) genotypes of L. angustifolius on the same or dissimilar seedling rootstocks. Fifteen seedlings of each cultivar were also harvested at this time by removing the shoot approximately 1 cm above the level of the soil. Xylem exudate was collected from the decapitated root system as above and stored at –20 °C. The seedlings were divided into roots and shoot, washed with deionized water, frozen (–20 °C) and then freeze-dried. Subsequent harvests of grafted plants, as well as control plants of each genotype, were made in later vegetative growth (49 DAS). Five plants of each graft type were harvested, divided into roots and shoots, washed with deionized water, immediately frozen (–20 °C) and then freeze-dried. Grafted plants not harvested were grown to physiological maturity and seed collected. Phloem exudate was collected from pod stalks every 7 d as above and stored at –20 °C.
Deuterated QA feeding experiments
Usually shoots bearing six fruits from either L. angustifolius or L. albus plants were cut from the main stem while the lower half of the plant was submerged under water. Taking care that the cut end of the shoot remained wet it was transferred to a 30 ml vial containing 20 ml of a 1 mg ml–1 aqueous solution of either D5-13-hydroxylupanine or D1-lupanine. The plants were left to transpire in a greenhouse and when most of the liquid had been taken up the vial was refilled with deionized water. After approximately 7 h phloem exudates were collected from leaf petioles and pod tips. Half the plants were divided into component organs and, after freezing (–20 °C), freeze-dried. The remaining plants were left to transpire for a further 14 h at which time phloem was collected and the organs harvested and processed as those at 7 h.
Preparation of tissue extracts and exudates for QA analysis
Freeze-dried plant material was finely ground at room temperature and 15 ml 5% (w/v) trichloroacetic acid added to 200 mg plant material. The suspension was tumbled at room temperature for 2 h followed by centrifugation at 3000 rpm for 15 min. A 12 ml aliquot of the supernatant was subsequently alkalinized with 25% (v/v) ammonia to pH 
11 and extracted twice with 25 ml dichloromethane. The pH was then raised to 14 by the addition of 10 M sodium hydroxide and again the solution extracted twice with 25 ml dichloromethane. The organic extracts were dried over anhydrous sodium sulphate, collected in a flask containing 100 µg of internal standard (n-eicosane) and concentrated in vacuo. The residues were reconstituted in c. 1 ml ethyl acetate.
Usually 50 µl phloem sap or 300 µl xylem sap were made up to 1 ml with 25% (v/v) ammonia. The aqueous solution was extracted four times with methylene chloride (2 ml) and the combined organic extracts dried over anhydrous sodium sulphate and concentrated with heating (40 °C) under a continuous stream of nitrogen. The residue was reconstituted in methanol (100 µl) containing caffeine (10 µg) as internal standard.
Identification and quantification of QA
Extracts were separated on a DB ultra 1 fused silica capillary column (12.5 mx0.25 mm (internal diameter); film thickness 0.25 µm) with He as carrier gas (1 ml min–1; split ratio 1:20, injection port 290 °C) and detection with both flame ionization (300 °C), and nitrogen phosphorus detectors (270 °C). QA were chromatographed using an oven temperature program starting at 150 °C, 1 min isothermal, 12.0 °C min–1 to 300 °C, and finally 5 min isothermal.
For capillary GC-MS the following conditions were used: GC carrier gas He, DB ultra-1 fused silica capillary column (12.5 mx0.2 mm i.d.; film thickness 0.33 µm), injection port temperature 250 °C, split ratio 1:20. Temperature programming was initiated 120 °C, 2 min isothermal, 15 °C min–1 to 310 °C and finally 5 min isothermal at 300 °C. The GC-MS transfer line temperature was 310 °C to a Hewlett Packard 5970 MSD with electron energy at 70 eV. Individual QA were identified by retention indices comparing their mass spectra, produced at 70 eV, with published data (Wink, 1987a) and to spectra from authentic standards. Total QA values were the sum of the individual QA expressed on a dry weight tissue basis and for xylem and phloem on a volume basis.
Labelled D5-13-hydroxylupanine samples and standards were analysed as the trifluoroacetate derivative. Tissue and phloem exudate samples were extracted as above and the residues treated with triflouroacetic acid (100 µl) and pyridine (10 µl) at 80 °C for 1 h. The reagents were evaporated under a stream of nitrogen and the residues reconstituted in ethyl acetate containing n-eicosane (0.1 mg ml–1) as an internal standard. Samples were then analysed by GC/MS as above in selected ion monitoring mode. Ions monitored for each compound were as follows: D1-lupanine (137, 249), D5-13-hydroxylupanine (252, 141) and n-eicosane (57, 282). Labelled QA concentrations were calculated using the ratio of the internal standard: labelled QA peak area compared with a 5-point standard curve.
Preparation of deuterated QA
13-oxolupanine (511 mg, 1.95 mmol) was dissolved in deuterium oxide (D2O, 15 ml) and the pH adjusted to 14 by addition of sodium deuteroxide solution (40% w/v in D2O). The solution was stirred at room temperature and the reaction progress monitored by GC/MS. The observed molecular ion gradually rose from 262 amu to 266 amu and when no 262 ion was observable the reaction was deemed to be complete (approximately 48 h). The solution was then extracted with methylene chloride (3x30 ml). The combined extracts were dried over anhydrous sodium sulphate and concentrated in vacuo, resulting in a colourless oil (490 mg, 1.8 mmol). The 2H-labelled 13-oxolupanine (490 mg, 1.8 mmol) was dissolved in deutero methanol (CH3OD) (10 ml) and sodium borodeuteride (125 mg, 3.0 mmol) was added to the stirred solution. After 30 min deuterium oxide was added to the reaction to quench any unreacted sodium borodeuteride. The deutero methanol/water solution was evaporated to dryness in vacuo and the residue reconstituted in 2 M sodium hydroxide (50 ml), extracted with methylene chloride (3x100 ml) and dried over anhydrous sodium sulphate. The organic extracts were dried over anhydrous sodium sulphate and then removed in vacuo resulting colourless oil (450 mg, 1.67 mmol). GC/MS of the oil revealed the compound to be D5-13-hydroxylupanine with a molecular ion equivalent to 269 amu equal to an enrichment of 5 amu.
Deuterated lupanine was prepared by dissolving unlabelled lupanine (1.0 g, 4.032 mmol) in 50 ml dichloromethane and with stirring adding N-bromosuccinimide (825 mg, 4.65 mmol). The yellow solution was stirred for 10 min, extracted with aqueous sodium hydroxide (2 M, 4x15 ml), dried over anhydrous sodium sulphate and concentrated in vacuo. The residue was dissolved in deutero methanol (CH3OD, 15 ml). Sodium borodeuteride (625 mg, 42 mmol) was added to the warmed solution (40 °C) and stirred for 12 h. The methanol was removed in vacuo and the residue reconstituted in aqueous sodium hydroxide. This solution was extracted with methylene chloride (4x25 ml), which was subsequently dried over anhydrous sodium sulphate and concentrated in vacuo resulting in colourless oil (959 mg, 3.85 mmol). GC/MS analysis of the product revealed an increase in the molecular weight of lupanine by 1 amu.
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Content and spectrum of QA in bitter and sweet cultivars of L. albus
The total QA concentrations (i.e. the sum of the spectrum of QAs measured) for cv. Lupini and cv. Ultra plant parts and transport fluids, averaged over the entire study period of reproductive development, are summarized in Fig. 1A–D. Across all tissues cv. Lupini contained about 50 times more QA than cv. Ultra and this was also the case for the exudates analysed. In both cultivars QA concentration increased towards the apex of the plant in newly produced tissues, particularly in reproductive organs and in both cultivars roots and nodules showed the lowest concentrations of QAs. Xylem levels of QA were 10–30 times lower than those in phloem exudates collected at three sites on each cultivar.
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All 13 QA measured in L. albus were detected in each of the organs of both cultivars. The summary data in Figs 2 and 3 shows the proportional composition averaged across the period of reproductive development in each case. Even though all QA were detected, those, whose composition when averaged over all the plant parts was less than 1%, were grouped as ‘others’. In all tissues of cv. Lupini, lupanine comprised the majority of accumulated QA (Fig. 2A) especially in seeds. Other prominent QAs present in the bitter cultivar were 13-hydroxylupanine and its esterified derivatives, in particular 13-tigloyloxylupanine. Although the seeds were not separated into component tissues, the liquid endosperm, which forms when the testa expands and before the embryo begins to ‘fill’, was collected separately and analysed for QA content (Fig. 3A). In cv. Lupini the total QAs averaged across collections of endosperm were 0.72±0.23 mg ml–1 (n=4) and, like the whole seed, comprised mainly lupanine with lesser concentrations of 13-tigloyloxylupanine. In cv. Ultra most tissues also contained lupanine as the major QA, but in reproductive tissues and seeds especially, 13-hydroxylupanine was prominent (Fig. 2B). The structural isomer,
-isolupanine, was present at levels less than 1% in the bitter cultivar, but was prominent in all tissues of cv. Ultra, comprising c. 20% of QA found in the below-ground organs and vegetation towards the base of the plant. In all, five esterified derivatives of 13-hydroxylupanine as well as one esterified derivative of 13-hydroxymultiflorine were detected in tissues of both cultivars.
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All the QA found in tissue extracts were represented in xylem and phloem exudates from both cultivars (Fig. 3A, B), and, like tissues, exudates contained lupanine and its hydroxy derivative as major components. Among the esters 13-tigloyloxylupanine was prominent while
-isolupanine was detectable in all samples but was not as prominent in exudates from cv. Ultra as in tissues of this cultivar.
Grafting experiments
The rate of recovery of successful grafts was much lower with bitter and sweet cultivars of L. albus than with L. angustifolius. Consequently, data from grafting experiments are confined to bitter (cv. Fest) and sweet (cv. Danja) cultivars of the latter.
Preliminary studies in which a series of homo grafts (i.e. Fest scions to Fest rootstocks and likewise for Danja) were established at 21 DAS, and their QA contents 28 d later (49 DAS), compared with similarly aged plants of each cultivar that had remained intact (i.e. not grafted) indicated that grafting had a small but statistically significant effect on the QA content and spectrum of individual alkaloids (Table 1). Grafting increased the total QA contents in the shoots of both cultivars by up to 2-fold with smaller, non-significant, effects on the total QA content of roots and phloem exudate. In general, all of the 13 individual QA measured in these samples increased by the same proportion. As a result of these subtle effects of grafting per se on QA contents of shoots and roots, the impact of hetero grafts was assessed by comparison with homografted plants prepared at the same time as the dissimilar rootstocks and scions were prepared.
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Danja shoots became enriched 17-fold in QA when grafted onto Fest rootstocks for 28 d in comparison to Danja shoots grafted onto their own rootstocks (Table 2). Except for lupanine and its structural isomer all other QA measured increased markedly, especially 13-hydroxylupanine and angustifoline and some of the esterified QA (e.g. 13-trans-cinnamoyloxylupanine). Fest shoots grafted onto Danja rootstocks showed a relatively small, but significant, reduction in total QA compared with Fest shoots grafted onto their own rootstocks (Table 2). Although there was a uniform reduction in all unesterified QA, this was not the case for the suite of esterified QA. In some cases the Fest shoot contained more of these when supported by the sweet Danja root. For example, 13-tigloyloxylupanine increased more than 10-fold while others doubled or remained the same. On the other hand 13-oxolupanine decreased 20-fold. The rootstocks of the heterografted plants also showed some significant changes in QA levels (Table 3). The Fest rootstock grafted onto the Danja scion contained 150 times more QA than Danja roots and more than double the level in its homograft. The elevated QA were the major constituents, but lupanine increased 27-fold. Similarly, the Danja rootstock accumulated 60-fold more QA as a result of carrying a Fest scion than did the homografted Danja rootstock. The total QA in this heterografted Danja root was similar to the roots of the homografted Fest plants and, in general, the composition was similar except that rather more angustifoline accumulated in the Danja root along with 10-fold more 13-oxolupanine and 13-tigloyloxylupanine (Table 3).
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Grafting also had a marked impact on the QA content of phloem exudate collected from developing fruits during the period of their development (Table 4). The Fest rootstock increased alkaloid levels in phloem of the Danja shoot about 4 fold, due mainly to increases in angustifoline and especially 13-hydroxylupanine. On the other hand the Danja rootstock had negligible impact on total QA levels in phloem from the Fest shoot. There were small changes in the spectrum of QA particularly among the esterified alkaloids. As a consequence of having a Fest rootstock, mature Danja seeds from the heterografted plants contained about 30% more QA (Table 5). Almost all this increase was due to 13-hydroxylupanine. The Danja rootstock caused a small decrease in the total QA recovered in mature Fest seeds and, interestingly, caused a significant increase in the esters in seeds, particularly in the trans-isomer of cinnamoyloxylupanine (Table 5).
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Labelling studies
Both deuterated substrates were taken up through the transpiration stream of cut shoots and distributed to all shoot organs, including fruits, of both species (Figs 4, 5). A greater amount of labelled lupanine was recovered in vegetative tissues of the shoot compared with the hydroxy derivative and this was the case for both species. However, similar amounts of each were recovered in seeds. Both substrates were recovered in phloem exudates from each species which, in the case of L. albus, showed clear evidence of progressive labelling with time. Phloem was also more heavily labelled with lupanine compared with 13-hydroxylupanine. The distribution patterns were different for the two species but, in each case, stems and petioles accumulated more label than leaves and fruit tissues. The endosperm was collected from seeds in each case and while both labelled QA could be detected the levels were much lower than those of the whole seeds.
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Discussion |
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Although the bitter cultivar of L. albus contained roughly 50 times more QA than the sweet cultivar, in each, the greatest concentrations of QA were found in the upper shoot organs and particularly in flowers and fruit tissues. The difference in QA levels was reflected in both xylem and phloem exudates, and while these data do not indicate whether QA are synthesized primarily in leaves or the degree to which there is synthesis elsewhere in the plant, they suggest that distribution through processes of translocation is a major contributor to patterns of accumulation. While there are significant differences in the spectrum of QA formed in the bitter and sweet cultivars of L. albus, in each, lupanine and its hydroxylated derivative are major components accumulating in all tissues and in exudates from xylem and phloem. The generally accepted pathway for QA synthesis in lupins regards the two primary tetracyclic molecules, sparteine and lupanine, to be substrates for synthesis of all the tetracyclic derivatives. Thus, both the primary pathway products as well as their metabolites, including some of the esters, are loaded onto xylem and phloem and translocated. Loading of the two major QA, lupanine and 13–hydroxylupanine, onto phloem was confirmed using isotopically labelled substrates. Each was recovered in phloem exudates collected at petioles of source leaves or at the major sink, the fruits.
Analysis of phloem exudates in both bitter and sweet cultivars of L. angustifolius also showed significant levels of a range of QA in the translocation pathway. While the major QA in shoots, 13-hydroxylupanine, lupanine, and angustifoline, were prominent in phloem, the cinnamoyl esters for example, although significant in the leaves, were hardly loaded onto phloem. Similarly methylbutyryloxylupanine was only found in phloem in trace amounts compared with the supporting shoot tissues. The data thus indicated some degree of specificity in the loading of QA into phloem and, as a result, the composition of QA accumulating in seeds as they developed more closely matched phloem composition than that of the vegetative organs of the shoot. This was also indicated by the relative loading of the two deuterated substrates onto phloem of both L. angustifolius and L. albus; lupanine appearing at greater levels and more rapidly in phloem than 13–hydroxylupanine.
The data for L. albus permit an assessment of the relative contributions of in situ synthesis and translocation to QA accumulation in developing fruits. A previously published study (Pate et al., 1979) provides quantitative measures of the fluxes of C, N, and water to developing fruits of cv. Ultra which can be exploited to estimate flows of any translocated solute(s) whose concentrations in xylem and phloem are known. Similar data for the cytokinins found in white lupin were used recently to estimate in situ synthesis and translocated flux for this group of plant growth regulators to seeds (Emery et al., 2000). Using this approach, estimates for xylem and phloem delivery of QA to fruits have been generated and, by difference, the proportion of alkaloids formed in situ calculated (Table 6). The estimates show that both cultivars derived about half of their accumulated QA from translocation, with half presumed to be the result of synthesis in fruit/seeds. The water economy was determined for cv. Ultra and, strictly, the values might not apply to cv. Lupini. However, the growth rates and fruiting habit of the two varieties are similar under the same conditions of culture and while there may be small differences between cultivars in water use, a large difference is unlikely. These estimates do not prove that in situ synthesis of QA occurs in fruits and more direct experiments using labelled precursors together with studies of gene expression are needed. Nevertheless, taken at face value they suggest that one of the consequences of the mutation that provided the sweet genotype in L. albus was that overall synthesis was reduced both in vegetative and reproductive tissues and that translocation of QA reflected the level of synthesis.
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Thus attempts to breed a variety with a bitter vegetation/sweet seed phenotype would require significant changes to the mechanism(s) loading QA onto the translocation channels, particularly phloem, or to specific inhibition of QA synthesis in fruits. That phloem loading of QA appears to exhibit some degree of specificity might reflect differences in the affinity of transporters for the major QA substrates or the expression of a diversity of transporters with affinities for both major and less abundant QA. In either case, studies to characterize QA transporters and use transgenic techniques to alter their patterns of expression or specificity could well indicate how the potential for the ‘bitter/sweet’ phenotypes envisaged by Wink (1990a) might be generated. Whether maintaining a high level of QA in phloem is necessary for resistance to aphid feeding is not known. There is evidence that resistance to cowpea aphid (Aphis craccivora Koch) found in some lines of L. luteus and L. angustifolius is due in part to phloem–based deterrence (Zehnder et al., 2001), but whether alkaloid content or a particular QA species is responsible has not been established. Genotype-specific resistance induced by prior Myzus persicae feeding has been found in cultivars of both narrow-leafed and yellow lupin species (Cardoza et al., 2005) and, while causative factors were not identified, clearly a number of mechanisms contribute to deter attack.
The grafting experiments using bitter and sweet genotypes of L. angustifolius were designed to test the ideas put forward by Bäumel et al. (1995) that roots were the major site for QA synthesis, and that xylem translocation was the main route for QA distribution. Comparison of QA content between plants that were intact and plants that had been grafted with their own shoots indicated that grafting caused a significant increase in the shoots of both sweet and bitter varieties 28 d after the grafts were established. This response could be the result of an inductive response to damage and so accords well with other studies that showed wounding increased QA content (Wink, 1983, 1990b). Vilarino et al. (2005) have reported increases in QA following herbivore damage in both sweet and bitter cultivars of L. albus but, interestingly, no response in the L. angustifolius cultivars tested. However, the data indicate the necessity of comparing heterografts with homografted plants rather than with intact plants.
While the bitter rootstock caused a significant increase in QA levels of grafted Danja shoots the level was relatively small compared to those accumulating in Fest shoots whether grafted to their own or Danja rootstocks (Table 2). Interestingly, the presence of a sweet shoot caused a significant increase in the QA content of the bitter rootstock that supported the scion (Table 3) and no doubt this was a major reason for the increased levels of QA in the Danja shoot. The results do not permit a clear picture of the relative rates of synthesis in shoot and root organs as accumulated QA formed in situ or imported from elsewhere in the plants could not be assessed separately. Similarly the amounts exported, whether in xylem or phloem, could not be estimated from the data for shoots or roots. However, the level of accumulation in the mature seed is likely to reflect changes in the major sites of synthesis as a consequence of grafting and it is notable that seed developed on the Danja scion supported by the Fest rootstock accumulated little more QA than if the Danja rootstock was present (Table 5). Not surprisingly, the somewhat increased accumulation of QA in Danja scions on a Fest rootstock resulted in a significant increase in the total concentration of QA in phloem exudate (Table 4). However, the increased levels were relatively small compared with the phloem content from a Fest scion.
Taken together these data support neither idea put forward by Bäumel et al. (1995), but rather confirm earlier conclusions that QA are synthesized mainly in the aerial tissues of lupins and that phloem provides the major means for their distribution (Wink and Hartmann, 1981; Wink, 1987c; Babaoglu et al., 2004). There is no doubt that the substantial flux of water through the xylem transpiration stream could potentially redistribute a significant overall quantity of QA from the root system. However, whether the xylem-borne QA flux is due solely to synthesis in roots seems unlikely. The grafting data support the idea that there is some synthesis in roots but it seems probable that a large proportion leaving in xylem was translocated from the shoot in phloem. A preliminary study using 14C-labelled 13-hydroxylupanine supplied to mature leaves of intact L. albus (cv. Lupini) plants through a reverse flap effectively labelled phloem exudate collected at a number of sites. Although in each of four individually fed plants the majority of 14C was recovered from young leaves and stems above the fed leaf, a low level of label was found in the roots and in xylem exudate collected from the root system after the shoot was removed. More extensive labelling studies using a number of translocated QA together with analysis of expression of QA pathway genes are required to establish more clearly the sites and levels of alkaloid synthesis in lupins.
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Acknowledgements |
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This research was supported by the Grains Research and Development Corporation (GRDC) of Australia and by staff and facilities of the Chemistry Centre of Western Australia.
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