The Transfer of Organic Substances from Host (Alnus glutinosa Gaertn) to the Holoparasitic Plant (Lathraea clandestina L.)
S. RENAUDIN*) and F. LARHER**)
*) Laboratoire de Cytopathologie Vegetale, Institut des Sciences de la Nature, Universite de Nantes, 2 rue de la Houssiniere, 44072 Nantes Cedex, France
**) Laboratoire de Biologie Vegetale, U.E.R. S.V.E., Universite de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France
Received March 2, 1981 . Accepted May 25, 1981
Summary
The host parasite association »Alnus glutinosa - Lathraea clandestina« was fed with HC02
and the organs of the two plants were then harvested. The incorporation of HC in the constituents of the different plant tissues (soluble and insoluble fractions) was then studied. The results obtained show large transfers of glutamic acid, citrulline and sucrose from the host plant to the parasite. Certain results suggest that Lathraea fixes CO2 by phosphoenol pyruvate carboxylase.
Key words: Lathraea clandestina, Alnus glutinosa, root parasite, metabolites, glutamate, citrulline, sucrose, PEP carboxylase.
Introduction
The parasitic Phanerogames derive a part of their nutritional requirements in water, mineral elements and organic substances from a neighbouring chlorophyll (green) plant. According to their needs for the carbon substrates provided by the host plant, parasites can be grouped into Holoparasites which are totally dependent on the host because of their lack of chlorophyll or Hemiparasites which are capable of photosynthesis and therefore not completely dependent on the host. However, the nature and importance of the transfer of organic substances which occurs between host plant and parasite have only been studied in a limited number of hemiparasites (Okonkwo, 1966; Govier, 1966; Govier et aI., 1967; Gouws et aI., 1980) and only in one holoparasite dodder which is a parasite of Vicia faba or Pelargonium sp. (W olswinkel, 1974; Fer, 1979).
In this study we looked at certain aspects of the transfer of radioactive substances which occur between the host plant fed with Heo2 and the root holoparasite.
*~ Permanent adress: Laboratoire de Biologie vegetale II, Faculte des Sciences, Centre de 2e cycle, Universite de Nancy I, 54037 Nancy Cedex_
Z. Pjlanzenphysiol. Ed. 104. S. 71- 80. 1981.
72 S. RENAUDIN and F. LARHER
Material and Methods
Lathraea obtained from pure culture (Renaudin, 1974) and Alnus plants both one year old are placed in contact and the association host-parasite is grown in pots of 7 cm diameter, the para-
Stems
Leave.
Nodule •• '. '" Roots. 'e. ,. ".\
••••••• "'~ . ::' .... :":::::-·::::·:··:~.-;-·::.-; .. : .. 7 .... \~.~.~~~~~~~.::~:".::: ..}
St~ms \\ /,' \-. :'
Perchloric
••••••••.•. acid
AERIAL
SUBTERRANEAN
Scale leaye. •• ••••••• ...... •• :,' \', !, ................... Plastic layer
Roots........ . ••••••• ~::'.. .:,' \", /:'
Haultorla ••••••••••••• \"'" •••• Aluminium '. f.··················· ····sh •• t \:' ~ ~':'::.' ~.::. ~:":' '::.' ~ '::.' ;'::. ':,
Fig. 1: Schematic representation of the experimental apparatus and of the host-parasite complex where only leaves and stems of alder were exposed to I'C02 and illuminated for 14 hours.
site being completly buried in the soil. For the experiment, the roots and nodules of Alnus and the Lathraea are isolated by a double protection system which consists of a layer of plastic covered by a sheet of aluminium paper (Fig. 1). The plants are then enclosed in a glass vessel containing HC02 at a concentration of 1 % at t = 0 and the temperature is kept at 20°C. HC02 is obtained by the addition of perchloric acid to a solution of Ba HC03; Alder is illuminated for 14 h and then placed in complete darkness for a period of 10 h. At the end of the treatment the host and parasite are separated, the organs isolated, fixed by liquid nitrogen and lyophillized. In the case of the host, the leaves, stems, roots without nodules or haustoria, and nodules are harvested and for the parasite, the roots without haustoria and the stolons with their scale leaves.
The samples are ground in a mortar and extracted using a water-alcohol mixture at 4 0C. The
Z. Pjlanzenphysiol. Bd. 104. S. 11-80. 1981.
Transfer of metabolites between two partners 73
methods of extraction, fractionation and separation as well as the method for measuring radioactivity were previously described by (Goas et al., 1970; Larher, 1976). The determination of ureides is carried out according to the method of Le Rudulier (1978); urea is isolated from the amino acid and free amide fraction by two dimensional chromatography.
The total radioactivity of the tissues and compounds isolated is given in 10-Jdpm . g-l dry matter and also in percentage of the total radioactivity of the studied samples. In the case of amino acids liberated by the hydrolysis of proteins the values are given in 10-Jdpm . g-l dry matter and in percentages of the total radioactivity of these substances.
Results
1. Incorporation of Radio-carbon in the Plant Tissues a) Organs of the Host Plant
In assimilating leaves, the assimilation of 14C02 results in an intense labelling of soluble carbohydrates (almost half of the total radioactivity TR; table 1). The incor-
Table 1: Distribution of radiocarbon in the studied tissues. Results are given in dpm x 1O-3/g dry matter (I) and % of total radioactivity TR (II).
Compounds Alnus glutinosa Lathraea clandestina studied Leaves Stems Roots Nodules Roots Stems
II II II II II
Soluble carbohydrates 902893 48.1 125197 59.2 127266 62.1 14807 27.7 4395 37.0 12554 Organic acids 103611 5.5 5698 2.7 4264 2.1 4949 9.3 875 7.4 6648 Free amino acids 18393 1.0 7690 3.6 10024 4.9 20800 38.9 1781 15.0 33541 U rea and ureides 1105 0.06 167 0.08 238 0.1 151 0.3 208 1.7 934 Protein amino acids 155167 8.3 5512 2.6 8559 4.2 2908 5.4 1172 9.9 3265 Non-protein insoluble fraction 695783 37.1 67316 31.8 54645 26.6 9781 18.4 3455 29.0 8038 Total radio-activity (TR) 1876952 211580 204996 53396 11866 64980
poration of 14C in the insoluble non-protein constituents is also high. Amongst the other fractions exhibiting radioactivity one can note the organic acids (5 % TR), the free amino acids (1 %) and the protein substances (8.3 % TR). The ureides are only feebly marked. In stems, the radioactivity found in the soluble carbohydrate fraction is very high (59 %) even more than in insoluble non-protein compounds. Amongst the soluble constituents free amino acids are prominent (3.6 % TR). In roots, the distribution is more or less identical with that observed in the case of stems, the relative significance of the labelling of carbohydrates and soluble amino acids being greater. In nodules, the distribution of radioactivity is different. The free amino acids incorporate the major part of 14e (39 % TR) whereas soluble carbohydrates and insoluble non-protein compounds are also well labelled. The intensity of the labelling of organic acids and ureides (9.3 and 0.3 % TR) is more significant than in all other organs.
Z. Pjlanzenphysiol. Bd. 104. S. 71-80. 1981.
II
19.3 10.2 51.8
1.4
5.0
12.3
74 S. RENAUDIN and F. LARHER
b) Organs of the Parasitic Plant
In roots in direct relation with the root system of the host plant, soluble carbohydrates showing 37 % TR appear to be the best labelled compounds; the labelling is even superior to that of the insoluble non-protein compounds. The radioactivity of the free amino acids (15 % TR) is significant and that of the ureides is greater than the value observed in the host plant. The level of radioactivity of the protein amino acids and organic acids remains appreciable. In non-chlorophyllous stems the labelling of free amino acids is significant (this accounts for 50 % TR). The radioactivity of soluble carbohydrates (19 % TR) as well as of non-protein insoluble constituents is also significant. It should be noted that the intensity of the labelling of free amino acids, organic acids and urea-ureides of these organs is superior to that of nodule tissue of Alnus.
For the organs of the two plants it is possible to calculate the relation (R) between the level of radioactivity of the non-nitrogen compounds and that of the nitrogen compounds. In the case of Alder, as one might expect, R is high in leaves, stems and roots (9.7, 14.9 and 9.9 respectively). R is only 1.2 in nodules where the rapid utilization of carbohydrates permits the fixation of nitrogen by nitrogenase and subsequent incorporation of ammonia in organic molecules. In Lathraea, the significance of the R values is low. In roots (R = 2.8), carbohydrates remain predominant but in stems where R = 0.7 nitrogen compounds are preponderant. Results obtained at an equilibrium point do not permit the estimation of the transformations carried out by the parasitic plant. In any case, the ratios suggest the possible existence of a transfer of radiocarbon from the host plant to the parasite in the form of soluble nitrogen compounds.
2. The degree of labelling of the compounds isolated by chromatography and electrophoresis
a) Soluble Compounds (Table 2)
Organs of the Host Plant
Soluble carbohydrates. In all organs the major part of the He is found incorporated in glucose, fructose and sucrose. Sucrose alone accounts for almost a quarter of the total radioactivity in leaves and almost half in stems and roots (transport organs and organs to which the products of photosynthesis are designated). In nodules, again sucrose accounts for 17 % TR. The glucose-fructose couple is relatively better labelled in assimilating organs than in other organs.
Organic acids. In leaves, quinic acid is by far the most radioactive and is superior to malic and citric acids; succinic, fumaric and glycolic acids are also labelled. In stems and roots, malic and citric acids are by far the most radioactive. The same holds in the
Z. Pjlanzenphysiol. Bd. 104. S. 71-80. 1981.
Transfer of metabolites between two partners 75
Table 2: Radioactivity of compounds isolated from the soluble fraction of tissues. Results are given in dpm X 1O-3/g dry matter (I) and % of total radioactivity (TR) (II).
Compounds studied
~ Raffinose ~ Sucrose
} Glucose ~ fructose 5 Arabinose ~ Rhamnose ::2 Xylose a Total
Citric -a Succinic '2 Fumaric
'E Malic 20 Glycolic o Quinic
Total
~ Aspartic
~ Asparagine ~ Glutamic
...t Glutamine 2 Alanine ;; Citrulline ~ Unknown ~ Ornithine .~ Arginine ..;: Total ~ Allantoi'ne
..iJ Allantoic acid '2 Urea
Leaves II
69830 3.7 463570 24.7 255971 13.6 101702 5.4
11820 0.7 902893 48.1
8803 1705 235
28711 1082
63015 103611
650 569
10855 1164
453 3938
599 63
102 18393
189 693 223
1105
0.4 0.1 0.01 1.5 0.06 3.3 5.5
0.03 0.03 06
0.06 0.02 0.2 003
1.0 0.01 0.04 0.01 0.06
Alnus glutinosa
Stems II
1868 0.9 97488 46.1 15349 7.2 10223 4.8
289 0.2 125197 59.2
1959 83 27
1924 49
1656 5698
440 64
1331 84 83
5188 440
16 44
7690 37
113 17
167
0.9 0.04 0.01 0.9 0.02 0.8 2.7
0.2 0.Q3
0.6 0.04 0.04 2.4 0.2 0.01 0.02 3.6 0.02 0.05 0.01 0.08
Roots II
101608 49.6 11172 5.4 14189 6.9
297 0.2 127266 62.1
1707 142 98
2579 9
89 4624
216 161
1404 368
96 6819
889 39 32
10024 17 90
131 238
0.8 0.07 0.05 1.3
0.04 2.2
0.1 0.08 0.7 0.2 0.05 3.3 0.4 0.02 0.01 4.9
0.04 0.06 0.1
850950 45.3 72828 34.4 63204 30.8
;:J Total Insoluble compound~
TR 1876952 211580 204996
Nodules II
9469 17.7 3750 7.0 1588 3.0
14807 27.7
1891 3.5 179 0.3
18 0.03 2664 5.0
143 0.3 54 0.1
4949 9.3
434 0.8 62 0.1
4059 7.6 61 0.1
103 0.2 15403 28.8
610 1.1 33 0.06 38 0.07
20800 38.9 27 0.05 82 0.15 42 0.08
151 0.3
12689 23.8 53396
Lathraea clandestina
Roots II
3771 31.8 292 2.4 201 1. 7
71 0.6 60 0.6
4395 37.0
183 68 37
535 15 37
875
206 59
747 285
30 423
12 19
1781 42
114 52
208
1.5 0.6 0.3 4.5 0.1 0.3 7.4
1.7 0.5 6.3 2.4 0.2 3.6
0.1 0.1
15.0 0.3 0.9 0.5 1.7
4627 39.0 11866
Stems II
10898 16.8 608 0.9 311 0.5 695 1.0
42 0.06
12554 19.3
927 1.4 658 1.0 211 0.3
4649 7.2 136 0.2 67 0.1
6648 10.2
3511 5.3 207 0.3
23109 35.5 2740 4.2
323 0.5 3232 5.0
134 0.2 579 0.8
33541 51.R 26 0.04
113 0.17 795 1.2 934 1.4
11309 17.4 64980
case of nodules where malic acid alone incorporated the same amount of HC as all the other organic acids put together (5 % TR).
Amino acids and free am ides. In all organs of the host plant, with the exception of the leaves, citrulline is the most radioactive among the soluble nitrogen-containing compounds (0.2, 2.4, 3.3, 28.8 % of total radioactivity respectively for leaves, stems, roots and nodules). The most highly labelled substance in leaves is glutamic acid (0.6 % TR). This constituent is also significantly labelled in nodules (7.6 % TR).
Urea, ureides. In all organs the quantity of HC incorporated in these compounds remains insignificant. At most it is 0.3 % TR in the nodules.
Organs of the Parasitic Plant
Soluble carbohydrates. In roots the preponderance of sucrose is shown by its degree
Z. Pjlanzenphysiol. Bd. 104. S. 71-80. 1981.
76 S. RENAUDIN and F. LARHER
of labelling 31.8 % TR; in stems it is also well labelled (16.8 % TR). Glucose and fructose show insignificant radioactivity.
Organic acids. Labelling of organic acids is relatively stronger in Lathraea than in the host plant. In roots, the 14C of malate represents more than half of the 14C of all the acids and as much as 10 % in stems where the diacid contains 7.2 % TR.
Amino acids and free am ides. The high radioactivity exhibited by the free amino acids is due to glutamic acid in roots (6.3 % TR) as well as in stem (35.5 % TR). The presence of radioactive citrulline is also noticeable (3.6 % TR in roots and 5 % in stems). Lastly, aspartic acid is notable in stems (5.3 % TR). Of the two amides glutamine is the best labelled and in stems it contains 4.2 % HC of the total radioactivity of the components.
Urea and ureides. These nitrogen rich components are more radioactive in the parasitic plant than in the host plant. In roots allantoic acid is the best labelled and in stems it is urea.
b) Protein Amino Acids (Table 3)
All protein amino acids are radioactive; generally speaking, glutamic acid and its amide incorporated most of the radio-carbon. This is very clear in the case of the
nodules of Alnus, the site of production of these two compounds, and also in the
Table 3: Radioactivity of protein amino acids. Results are given in dpm X JO-3/g dry matter (a) and % of total radioactivity of proteins (b).
Protein Alnus glutinosa Lathraea clandestina Amino Leaves Stems Roots Nodules Roots Stems Acids b b b b b b
Aspartic + Asparagine 15462 9.9 487 8.8 1308 15.3 635 21.8 156 13.3 596 18.2 Glutamic + Glutamine 16752 10.8 1188 21.5 2337 27.3 994 34.2 226 19.3 961 29.4 Serine 23039 14.9 410 7.4 443 5.2 146 5.0 29 2.5 56 1.7 Glycine 15557 10.0 388 7.0 253 2.9 82 2.8 21 1.8 27 0.8 Threonine 4477 2.9 143 2.6 238 2.8 54 1.8 21 1.8 45 1.4 Alanine 13514 8.7 259 4.7 626 7.3 330 11.3 25 2.1 112 3.4 Tyrosine 7541 4.8 188 3.4 167 1.9 52 1.8 16 0.5 Lysine 3907 2.5 312 5.6 358 4.2 31 1.1 29 2.5 87 2.7 Histidine 3143 2.0 273 4.9 518 6.0 71 2.4 316 26.9 795 24.3 Arginine 6624 42 436 7.9 472 5.5 40 1.4 132 11.3 274 8.4 Proline 3625 2.3 334 6.0 479 5.6 ISS 5.3 64 5.4 156 4.8 Valine + Methionine 9302 6.0 278 5.0 325 3.8 133 4.6 12 1.0 63 1.9 Leucine + Isoleucine + Phenylalanine 32204 20.9 816 14.8 817 9.5 138 4.7 141 12.0 77 2.3 Hydroxyproline 218 2.5 47 1.6 Total radioactivity of protein 155167 5512 8559 2908 1172 3265
Z. Pjlanzenphysiol. Bd. 104. S. 71-80. 1981.
Transfer of metabolites between two partners 77
stems of Lathraea in which we have already indicated the importance of free glutamate. Other amino acids are well marked: In Alnus: Serine in the leaves, and all the aspartic acid and asparagine in roots and nodules. In Lathraea: All the aspartic acid and asparagine, histidine and arginine.
Discussion
Under the experimental conditions used, maintaining the association Alnus gluti· nosa-Lathraea clandestina in an illuminated atmosphere enriched in HCOz results in a significant labelling of the constituents of the different organs of the parasitic plant. This is achieved by means of the haustoria fixed on the roots of Alnus which fed on the metabolites elaborated by the host. The significance of this uptake of metabolites has been shwon equally in the case of dodder, another holoparasite which is associated either with Vicia/aba Oacob, 1968; Jacob and Neumann, 1968; Wolswinkel, 1974} or with Pelargonium (Fer, 1979). It seems to be necessary for plants incapable of achieving the photosynthetic fixation of COz. Nevertheless certain results suggest, in the case of Lathraea, the existence of a limited autotrophic activity with respect to carbon. In effect the significant labelling of malic and aspartic acids in stems and roots could reflect a reduction and a rapid amination of oxalo-acetic acid, this acid being formed by the action of a phosphoenol-pyruvate carboxylase. The activity of this enzyme has already been disclosed in non-chlorophyllic organs (De Vries et aI., 1980) and shown in the organs of another Angiosperm parasite Orobanche lucorum (Soldatini et al., 1980). In these conditions it seems difficult to attribute a precise origin to malic and aspartic acids isolated from the different organs of Lathraea clandestina. In the case of soluble carbohydrates where labelling in the root of Lathraea represents 37 % of the total radioactivity and 60 % of the radioactivity of all the soluble compounds, one can imagine a possible important transfer of sucrose. Sucrose contains almost 1/3 of the total radioactivity and is again well labelled in stems. Similar data have been reported for dodder, a parasite of Pelargonium (Fer, 1979), and in Cuscuta pentagona (Kerstetter and Hull, 1970). In these cases, however, there is no structural obstacle to the transfer of sucrose because the parasite directly withdraws metabolites from the host sieve tubes through its absorbing hypha which show special structures in their plasmalemma and cell walls (Dorr, 1968), and sucrose is particularly abundant in phloem exsudates (Ziegler, 1975). In the case of Lathraea, the situation is much more complicated because in the haustoria, cellulosic absorbing cells (digitate cells, Kuijt, 1977) are separated from the phloem of the host by a lignin rich disorganized zone which prevents the transfer of metabolites (Renaudin, 1974). The best way for transport is the lignified bridge which is branched on the host vessels (Renaudin, 1974). Thus the parasite may obtain sucrose from the bleeding sap (Ziegler, 1955) of the Alder, since in woody species the sucrose content of xylem fluid is much higher than in herbaceous plants (Pate, 1976).
Z. Pjlanzenphysiol. Ed. 104. S. 71-80. 1981.
78 S. RENAUDIN and F. LARHER
Among other labelled metabolites, free amino acids are important particularly in stems where they represent 1/2 of the total radioactivity. The question can be asked wether the transfers take place from the host or wether there is a biosynthesis in situ from the 14C precursors onwards. In effect, the work of Govier et ai. (1967) suggests that in the case of Odontites verna there is an important flow of amino acids from the host to the parasite especially when the latter is fixed on root nodules of a Leguminous plant e. g. Trifolium. The results of Srivastava and Chauhan (1977) concerning dodder and those of Stewart and Orebamjo (1980) on Tapinanthus bang;wensis showed the presence in the parasitic plant not only of a nitrate and nitrite reducing enzyme but also of the enzyme involved in ammonia assimilation: glutamine synthetase. In the association studied, the host plant formed root nodules with Frankia. In these organs the utilisation of 14C permits significant labelling of citrulline (28 % TR of organs) and glutamic acid (7.6 %). It is a well known fact that these two substances are major amino acids in the nodules of Alder and that citrulline transported by the xylem represents the form in which nitrogen circulates in this species (Wheeler and Bond, 1970; Akkermanns et aI., 1979). Citrulline is found labelled not only in all organs of the host plant but also in those of the plant parasite. Although the specific patterns of nitrogen metabolism in Lathraea are unknown, it is quite plausible to think that citrulline synthesized in the nodules of Alder is easily directed to the parasite as it is transported in the xylem, the system from which Lathraea draws its nutrition.
Citrulline seems to be metabolised in the organs of Lathraea because arginine and ornithine, two other intermediate compounds in the Krebs-Henseleit cycle, are also labelled. Under these conditions the nitrogen of the molecule.can be free and used again later for the synthesis of glutamate and other amino acids. The carbon skeleton can be reused in this synthesis. A direct transfer of glutamic acid from the host to the parasite is also considered. Lathraea will have at its disposition an ideal substrate catabolism of which can get free ammonia, NADH2 and ketoglutaric acid directly reemployed or degraded in the Krebs' cycle.
As regards urea and ureides, these are found labelled in all organs of the two plants but labelling is more significant in Lathraea. The hypothesis of a possible synthesis in the nodules of Alnus leads one to think of preferential transport in the direction of the parasite. However, these compounds represent only a small part of the radioactivity transfered and their contribution to the nutrition of Lathraea appears unimportant. The duration of the experiment being 24 h labelled allantoine and allantoic acid simply correspond to pure catabolism and that of urea to the degradation of arginine formed from citrulline.
The incorporation of radioactivity in the insoluble compounds of the parasitic plant appears relatively higher in roots (39 % TR) than in stems (17.4 % TR). In the former part of the 14C can be found in starch since it is known that certain root tissues of Lathraea are particularly rich in amyloplasts (Renaudin, 1974). Whatever the case might be, one can say that the radioactive substrates withdrawn from the host plant by the haustoria are rapidly utilized for the biosynthesis of the macromolocules
z. Pjlanzenphysiol. Bd. 104. S. 71-80. 1981.
Transfer of metabolites between two partners 79
of structure and reserve tissues, whether or not they are previously transformed by Lathraea.
Only the radioactivity of the amino acids arising from the hydrolysis of proteins is determined. Significant labelling of aspartic and glutamic acids correlates well with the high radioactivity of these substances in their free state. The high figure observed for arginine is explained by its rapid biosynthesis from citrulline of the host plant via the Krebs-Henseleit cycle.
This cycle probably exists in Lathraea and Alnus, judging from the presence of radioactive citrulline, arginine and ornithine in all organs. The high intensity of labelling of histidine obtained from the hydrolysis of proteins remains unexplained. This might originate from a specific metabolic pathway in the parasitic plant since in the host plant the radioactivity of histidine is not significant.
Conclusion
During the utilisation of 14C02 by Alnus glutinosa a great number of radioactive compounds appear in the tissues of the parasite Lathraea clandestina suggesting possible transfers from host to parasite. A preferential holoparasitic relationship is manifested for soluble nitrogen compounds, particularly glutamic acid and citrulline. These compounds synthesized in the nodules of Alnus, are transported by the xylem system towards the consuming organs. Amongst these organs Lathraea seems to be a privileged addressee thanks to its haustorial system which withdraws metabolites directly from the xylem system of Alnus. The parasitic plant is also capable of obtaining sucrose previously recycled from the phloem. Lathraea can also achieve an anaplerotic fixation of carbon dioxide using the enzyme phosphoenol-pyruvate carboxylase. This permits rapid formation of malic and aspartic acids. We intend to characterize this enzyme and to determine its activity in Lathraea clandestina in order to discover its precise role in carbon nutrition of this Angiosperm parasite.
Acknowledgements
The excellent technical assistance of Mrs. A. Moreau, MM. Sealy L., Hervochon P. and Boursier M. is very gratefully acknowledged.
References
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