The Transfer of Organic Substances from Host (Alnus glutinosa Gaertn) to the Holoparasitic Plant (Lathraea dandestina L.)

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<ul><li><p>The Transfer of Organic Substances from Host (Alnus glutinosa Gaertn) to the Holoparasitic Plant (Lathraea clandestina L.) S. RENAUDIN*) and F. LARHER**) </p><p>*) Laboratoire de Cytopathologie Vegetale, Institut des Sciences de la Nature, Universite de Nantes, 2 rue de la Houssiniere, 44072 Nantes Cedex, France </p><p>**) Laboratoire de Biologie Vegetale, U.E.R. S.V.E., Universite de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France </p><p>Received March 2, 1981 . Accepted May 25, 1981 </p><p>Summary The host parasite association Alnus glutinosa - Lathraea clandestina was fed with HC02 </p><p>and the organs of the two plants were then harvested. The incorporation of HC in the consti-tuents 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 car-boxylase. </p><p>Key words: Lathraea clandestina, Alnus glutinosa, root parasite, metabolites, glutamate, citrul-line, sucrose, PEP carboxylase. </p><p>Introduction </p><p>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 ols-winkel, 1974; Fer, 1979). </p><p>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. </p><p>*~ Permanent adress: Laboratoire de Biologie vegetale II, Faculte des Sciences, Centre de 2e cycle, Universite de Nancy I, 54037 Nancy Cedex_ </p><p>Z. Pjlanzenphysiol. Ed. 104. S. 71- 80. 1981. </p></li><li><p>72 S. RENAUDIN and F. LARHER </p><p>Material and Methods Lathraea obtained from pure culture (Renaudin, 1974) and Alnus plants both one year old are </p><p>placed in contact and the association host-parasite is grown in pots of 7 cm diameter, the para-</p><p>Stems </p><p>Leave. </p><p>Nodule '. </p><p>'" Roots. 'e. ,. ".\ "'~ . ::' .... :":::::-::::::~.-;-::.-; .. : .. 7 </p><p>.... \~.~.~~~~~~~.::~:".::: ..} St~ms \\ /,' \-. :' </p><p>Perchloric .. acid </p><p>AERIAL </p><p>SUBTERRANEAN </p><p>Scale leaye. ...... :,' \', !, ................... Plastic layer </p><p>Roots........ . ~::'.. .:,' \", /:' </p><p>Haultorla \"'" Aluminium '. f. sh t \:' ~ ~':'::.' ~.::. ~:":' '::.' ~ '::.' ;'::. ':, </p><p>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. </p><p>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 cov-ered by a sheet of aluminium paper (Fig. 1). The plants are then enclosed in a glass vessel con-taining HC02 at a concentration of 1 % at t = 0 and the temperature is kept at 20C. 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 harv-ested and for the parasite, the roots without haustoria and the stolons with their scale leaves. </p><p>The samples are ground in a mortar and extracted using a water-alcohol mixture at 4 0C. The </p><p>Z. Pjlanzenphysiol. Bd. 104. S. 11-80. 1981. </p></li><li><p>Transfer of metabolites between two partners 73 </p><p>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. </p><p>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 sampl-es. 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. </p><p>Results </p><p>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 </p><p>soluble carbohydrates (almost half of the total radioactivity TR; table 1). The incor-</p><p>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 </p><p>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 </p><p>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 dis-tribution 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 incorpo-rate 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 orga-nic acids and ureides (9.3 and 0.3 % TR) is more significant than in all other organs. </p><p>Z. Pjlanzenphysiol. Bd. 104. S. 71-80. 1981. </p><p>II </p><p>19.3 10.2 51.8 </p><p>1.4 </p><p>5.0 </p><p>12.3 </p></li><li><p>74 S. RENAUDIN and F. LARHER </p><p>b) Organs of the Parasitic Plant In roots in direct relation with the root system of the host plant, soluble carbo-</p><p>hydrates 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 solu-ble 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. </p><p>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 utiliza-tion 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 equilib-rium 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 com-pounds. </p><p>2. The degree of labelling of the compounds isolated by chromatography and electrophoresis </p><p>a) Soluble Compounds (Table 2) Organs of the Host Plant Soluble carbohydrates. In all organs the major part of the He is found incorporated </p><p>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 suc-rose accounts for 17 % TR. The glucose-fructose couple is relatively better labelled in assimilating organs than in other organs. </p><p>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 </p><p>Z. Pjlanzenphysiol. Bd. 104. S. 71-80. 1981. </p></li><li><p>Transfer of metabolites between two partners 75 </p><p>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). </p><p>Compounds studied </p><p>~ Raffinose ~ Sucrose </p><p>} Glucose ~ fructose 5 Arabinose ~ Rhamnose ::2 Xylose a Total </p><p>Citric -a Succinic '2 Fumaric 'E Malic 20 Glycolic o Quinic </p><p>Total ~ Aspartic ~ Asparagine </p><p>~ Glutamic ...t Glutamine 2 Alanine ;; Citrulline ~ Unknown </p><p>~ Ornithine .~ Arginine ..;: Total </p><p>~ Allantoi'ne ..iJ Allantoic acid '2 Urea </p><p>Leaves II </p><p>69830 3.7 463570 24.7 255971 13.6 101702 5.4 </p><p>11820 0.7 902893 48.1 </p><p>8803 1705 235 </p><p>28711 1082 </p><p>63015 103611 </p><p>650 569 </p><p>10855 1164 </p><p>453 3938 </p><p>599 63 </p><p>102 18393 </p><p>189 693 223 </p><p>1105 </p><p>0.4 0.1 0.01 1.5 0.06 3.3 5.5 </p><p>0.03 0.03 06 </p><p>0.06 0.02 0.2 003 </p><p>1.0 0.01 0.04 0.01 0.06 </p><p>Alnus glutinosa </p><p>Stems II </p><p>1868 0.9 97488 46.1 15349 7.2 10223 4.8 </p><p>289 0.2 125197 59.2 </p><p>1959 83 27 </p><p>1924 49 </p><p>1656 5698 </p><p>440 64 </p><p>1331 84 83 </p><p>5188 440 </p><p>16 44 </p><p>7690 37 </p><p>113 17 </p><p>167 </p><p>0.9 0.04 0.01 0.9 0.02 0.8 2.7 </p><p>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 </p><p>Roots II </p><p>101608 49.6 11172 5.4 14189 6.9 </p><p>297 0.2 127266 62.1 </p><p>1707 142 98 </p><p>2579 9 </p><p>89 4624 </p><p>216 161 </p><p>1404 368 </p><p>96 6819 </p><p>889 39 32 </p><p>10024 17 90 </p><p>131 238 </p><p>0.8 0.07 0.05 1.3 </p><p>0.04 2.2 </p><p>0.1 0.08 0.7 0.2 0.05 3.3 0.4 0.02 0.01 4.9 </p><p>0.04 0.06 0.1 </p><p>850950 45.3 72828 34.4 63204 30.8 </p><p>;:J Total Insoluble compound~ TR 1876952 211580 204996 </p><p>Nodules II </p><p>9469 17.7 3750 7.0 1588 3.0 </p><p>14807 27.7 </p><p>1891 3.5 179 0.3 </p><p>18 0.03 2664 5.0 </p><p>143 0.3 54 0.1 </p><p>4949 9.3 </p><p>434 0.8 62 0.1 </p><p>4059 7.6 61 0.1 </p><p>103 0.2 15403 28.8 </p><p>610 1.1 33 0.06 38 0.07 </p><p>20800 38.9 27 0.05 82 0.15 42 0.08 </p><p>151 0.3 </p><p>12689 23.8 53396 </p><p>Lathraea clandestina </p><p>Roots II </p><p>3771 31.8 292 2.4 201 1. 7 </p><p>71 0.6 60 0.6 </p><p>4395 37.0 </p><p>183 68 37 </p><p>535 15 37 </p><p>875 </p><p>206 59 </p><p>747 285 </p><p>30 423 </p><p>12 19 </p><p>1781 42 </p><p>114 52 </p><p>208 </p><p>1.5 0.6 0.3 4.5 0.1 0.3 7.4 </p><p>1.7 0.5 6.3 2.4 0.2 3.6 </p><p>0.1 0.1 </p><p>15.0 0.3 0.9 0.5 1.7 </p><p>4627 39.0 11866 </p><p>Stems II </p><p>10898 16.8 608 0.9 311 0.5 695 1.0 </p><p>42 0.06 </p><p>12554 19.3 </p><p>927 1.4 658 1.0 211 0.3 </p><p>4649 7.2 136 0.2 67 0.1 </p><p>6648 10.2 </p><p>3511 5.3 207 0.3 </p><p>23109 35.5 2740 4.2 </p><p>323 0.5 3232 5.0 </p><p>134 0.2 579 0.8 </p><p>33541 51.R 26 0.04 </p><p>113 0.17 795 1.2 934 1.4 </p><p>11309 17.4 64980 </p><p>case of nodules where malic acid alone incorporated the same amount of HC as all the other organic acids put together (5 % TR). </p><p>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). </p><p>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. </p><p>Organs of the Parasitic Plant Soluble carbohydrates. In roots the preponderance of sucrose is shown by its degree </p><p>Z. Pjlanzenphysiol. Bd. 104. S. 71-80. 1981. </p></li><li><p>76 S. RENAUDIN and F. LARHER </p><p>of labelling 31.8 % TR; in stems it is also well labelled (16.8 % TR). Glucose and fruc-tose show insignificant radioactivity. </p><p>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. </p><p>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 gluta-mine is the best labelled and in stems it contains 4.2 % HC of the total radioactivity of the components. </p><p>Urea and ureides. These nitrogen rich components are more radioactive in the para-sitic plant than in the host plant. In roots allantoic acid is the best labelled and in stems it is urea. </p><p>b) Protein Amino Acids (Table 3) All protein amino acids are radioactive; generally speaking, glutamic acid and its </p><p>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 </p><p>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...</p></li></ul>

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