Solute fluxes from tobacco to the parasitic ... - Wiley Online Library

Plant, Cell and Environment (1999) 22, 937–947

Solute fluxes from tobacco to the parasitic angiosperm Orobanche cernua and the influence of infection on host carbon and nitrogen relations J. M. HIBBERD1, W. P. QUICK1, M. C. PRESS1, J. D. SCHOLES1 & W. D. JESCHKE2 1

Robert Hill Institute, Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN, UK and Julius-von-Sachs Institut für Biowissenschaften, Lehrstuhl für Botanik 1, Universität Würzburg, Mittlerer Dallenbergweg 64, D-97082, Würzburg, Germany

2

ABSTRACT Orobanche species are holoparasites which are very efficient sinks for host-derived solutes. Here, we report the use of direct measurements of xylem sap solute concentrations and water fluxes, together with a modelling procedure to calculate element fluxes within an association between Orobanche cernua and its tobacco host. Infection of tobacco by the parasite markedly influenced carbon acquisition and partitioning; net fixation of carbon was 20% higher in infected tobacco compared with controls. Orobanche cernua caused a 84% increase in net carbon flux moving downward from the tobacco shoot and 73% of this carbon was intercepted by the parasite, almost entirely >99%). Further, the parasite also through the phloem (> exerted a large impact on the nitrogen relations of the plant, notably nitrate uptake was stimulated and the amino acid content of xylem sap was lower. The parasite also relied heavily on host phloem for the supply of other resources, with only 5 to 15% of N, and 16% of K, 23% of Na, 63% of Mg and 13% of S being derived from the xylem. Thus, we provide quantitative information on the phloem dependency of the parasite and show that host carbon and nitrogen metabolism is stimulated as a consequence of infection. Key-words: Orobanche cernua; carbon; modelling; nitrogen; parasitic angiosperm; solute fluxes.

INTRODUCTION Plants that rely on others for the supply of nutrients are termed parasitic. They are found in at least 17 families (Parker & Riches 1993) and inhabit ecosystems ranging from the arctic to the tropics (Press 1998). In terms of their evolution, their ecological role and their physiology, parasitic plants represent an intriguing, but understudied group.

Correspondence: J. M. Hibberd. Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK. Fax 44 1223 333953; e-mail: [email protected] © 1999 Blackwell Science Ltd

For example, the Orobanchaceae have evolved such that photosynthetic ability is totally lost and the root system is only vestigial. Orobanche species are therefore totally reliant on their hosts for the supply of both reduced carbon and nitrogen, and also inorganic nutrients (Parker & Riches 1993). Relatively few studies have quantified how parasitism by Orobanche influences host growth, particularly with regard to the removal of resources. Hibberd et al. (1998) have recently used tobacco that was infected by Orobanche cernua to investigate the mechanisms by which this parasite influences host growth and the data suggested that lower rates of host growth can be directly attributed to removal of host assimilates by the parasitic sink. Remarkably, a smaller amount of photosynthetic biomass in the infected system is capable of maintaining a much larger weight of tissue that is capable of respiring, compared with uninfected tobacco plants. This was brought about by a combination of morphological and physiological changes, although their relative importance was not quantified. Our study suggested that infection resulted in a significant redirection of host assimilates from host sinks to the parasite, and efficient removal of assimilates by O. cernua. Orobanche withdraws nutrients from the host roots via an haustorium which forms close contacts with host vascular tissue (Kuijt & Toth 1976). Rates of transpiration of holoparasitic plants are generally very low compared with their hosts (Seel et al. 1992). Therefore, although direct links between xylem elements of the host and parasite have been reported (Kuijt & Toth 1976), the supply of solutes via the xylem to the parasite is likely to be low, because the low rate of parasite transpiration provides a small driving force for solute flux within the xylem. Pores derived from interspecific plasmodesmata connecting the host phloem with that of the parasite have also been reported (Dörr 1996) and indicate that nutrients may be derived directly from the host phloem. However, anatomical connections give no quantitative information on the flux through a pathway: we do not know how Orobanche removes assimilates from the host, nor do we know the route by which solutes are supplied to the parasite. 937

938 J. M. Hibberd et al. Using a similar rationale to that developed by Jeschke et al. (1994a) for the association between the shoot parasite Cuscuta reflexa and its host and, in addition, by directly sampling parasite xylem sap, we report nutrient flows within the infected tobacco and between tobacco and the parasite, O. cernua. The method was developed from the approach taken by Pate, Layzell & McNeil (1979) and Jeschke & Pate (1991). A similar partial modelling procedure has also been used to investigate flows of carbon and nitrogen between Acacia littorea and the root parasite Olax phyllanthi (Tennakoon, Pate & Fineran 1997). Both previous studies were able to estimate fluxes of solutes from host to parasite and the relative contribution that host xylem and phloem made in supplying those solutes. We aimed to estimate the importance of xylem in supplying solutes to O. cernua, and also the impact that infection has on flows of nutrients within the host. The experiments we report were designed to quantify the flux of solutes from the tobacco host to the parasite, and also the flows of elements within tobacco infected with O. cernua. Specifically, we aimed to test the following four hypotheses: (i) that infection increases the flux of host carbon towards the parasite; (ii) that the xylem does not represent the major supply route for solutes to the parasite; (iii) that the large amount of carbon removed from the host by the parasite will reduce recycling of carbon in the host xylem; and (iv) that there will be fewer carbon skeletons available to the root system for its growth, uptake of nutrients and reduction of nitrogen.

MATERIALS AND METHODS Growth of plants Seeds of tobacco were germinated and half of the plants inoculated as described in Hibberd et al. (1998). Parasite seed was placed onto the roots of the host so that the parasite would emerge about 2 cm away from the host stem. Up to day 24, tobacco plants were supplied with 2·2 mM nitrate in Rorison’s solution containing (in mM): NaCl, 1·6; Ca(NO3)2, 1·1; MgSO4, 1·0; K2HPO4, 1·01; CaCl2, 0·9; EDTA FeNa2, 0·07; H2SO4, 0·5; H3BO4, 0·05; MnSO4, 0·009; (NH4)6MO7O24, 0·00015; ZnSO4, 0·0015 and CuSO4, 0·0016. From then on, 4 mM nitrate was supplied by adding NaNO3, 1·65 mM and balancing Na and Ca with NaCl and CaCl2. Plants were grown from June to September in a greenhouse in Würzburg, Germany, with supplementary lighting to sustain minimum photon flux densities at 500 mmol m–2 s–1. Plants were grown under a 16 h light : 8 h dark photoperiod. Tobacco plants were grown in one of three types of pot: (a) pots designed to be placed in a pressure chamber so that xylem sap could be extracted; (b) airtight pots in which the roots could be sealed by placing silicon rubber, Blendascon (Blendamed Forschung, Bad Schwalbach, Germany) around the stems to allow measurements of root respiration; and (c) standard pots of 20 cm width for measurement of growth rates and the nutrient content of plant tissues.

Harvests of plant tissue for growth analysis, tissue gains in nutrients and the measurement of nitrate reductase activity At 38 and 49 d after half of the tobacco plants were inoculated with O. cernua seeds (62 to 73 d after tobacco seed germination), the plants were harvested. Plants within each treatment were paired prior to the harvests, and then separated into the root, the stem and the leaves, and, if plants were infected, O. cernua was separated from the root. All plant parts were weighed, freeze-dried, the dry tissue weighed and finely ground. Concentrations of C and N in dry matter were determined using a CHN analyser (CHNO-Rapid, Heräus, Hanau, Germany). The K, Mg, Na, Ca, P, and S were determined using an ICP spectrometer (JY 70 plus, ISA, Instrument S.A. Division, Jobin-Yvon, France). The content of each element in the organs and the increments over the study period were then calculated from the concentrations and the dry weights. Relative elemental increments (mmol mmol–1 d–1) and relative dry weight increments (g g–1 d–1) for the tobacco shoot and root and also the parasite were calculated over the study period. Between the two harvests at 38 and 49 d after inoculation with O. cernua, respiration of the host root, the host shoot and the parasite, and the concentration of amino acids, cations, anions and sugars in the xylem sap of host and parasite were measured daily. Additionally, at this time, the in vivo activity of nitrate reductase was determined after Andrews et al. (1992). Known weights of tissue were vacuum-infiltrated for 10 min in 10 cm3 of 100 mmol Na2HPO4/NaH2PO4 (pH 7·6) containing 50 mmol KNO3 and 3% (v/v) propan-1-ol.After vacuum infiltration, a 1 cm3 sample was taken as the zero-time point, and the remaining solution incubated, still under vacuum, in a shaking water bath at 30 °C for 20 min. After 20 min, another 1 cm3 sample was removed. The nitrite content of the samples was then determined at 543 nm, through comparison with nitrite standards. The absorbance was measured 10 min after the addition of 0·5 cm3 sulfhanalamide (1% v/v HCl) and 0·5 cm3 of N-naphthylenediamine. The difference between values from the zero time points, and final samples were taken as the nitrate reductase activity, and expressed as mmol NO2– g–1 fresh weight h–1.

Collection and analysis of xylem sap Xylem sap was collected between 38 and 49 d after inoculation, from the stem bases of tobacco and O. cernua. The sap was obtained by pressurizing the root system of plants placed in pots designed to fit into the pressure chamber (Passioura 1980). In order to collect sap from plants parasitized by O. cernua, and O. cernua itself, a special lid was designed and constructed for the pressure chamber. The lid had two openings, one for the host stem and the other for the O. cernua spikelet. Prior to enclosing the pot, excess water was drained from the sand by applying a vacuum and then the hypocotyl was sealed to the lid using Blendascon (see above). The root system was pressurized until sap © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 937–947

Withdrawal of tobacco nutrients by Orobanche 939 flowed from an incision made at the stem base of the tobacco (the mean balancing pressure was 0·38 MPa). Orobanche cernua only yielded xylem sap (at 0·25 MPa) after the shoot of the tobacco host had been excised and the root system and host stem resealed within the pressure chamber. Sap was stored at – 35 °C prior to analysis. Anions and cations were analysed (including nitrate, malate and oxalate) (Anionchromatograph IC 1000; EppendorfBiotronik, Maintal, Germany). Amino acids were estimated by the ninhydrin method (Amino Acid Analyser LC 5001; Eppendorf-Biotronik).

Assessment of gas exchange during the study period Plants established in sealable pots were used to assess the respiration of the roots of controls, the roots and associated underground biomass of the parasite in infected plants (Pate et al. 1979), and the night-time respiration of the shoots of the host (Jeschke et al. 1994a). Respiration and transpiration rates of above ground matter of the parasite while remaining attached to the host were assessed by enclosing the material in a chamber (20 cm length, 5 cm diameter) linked to an infra red gas analyser (IRGA) (LCA4; ADC, Hoddesdon, UK). Measurements were made at 15 min intervals over the 24 h photoperiod, and the average loss of carbon and water over the 24 h photoperiod was calculated.

Estimation of cross-sectional area of xylem vessels and resistance to flows The segments of tobacco and O. cernua stem that were excised in order to collect xylem sap were sectioned transversely and photomicrographs taken. The diameter of individual xylem elements was measured with a microscope and the mean cross-sectional area of xylem vessels in the two species calculated. The pressure difference required to move xylem sap a distance of 6 cm (the length of the O. cernua and tobacco stems to the point where the sap was collected) through the measured cross-sectional area of vessels, was calculated using the Hagen-Poiseuille equation.

Calculation of solute flows within the host and between host and parasite Based on the assumption that mass flow occurred in the xylem between host root and shoot, the net flow of each element along this route was calculated. By sampling xylem sap of the parasite, we were able to also investigate specificity in the transport of solutes within the parasite xylem. The flux of each nutrient supplied by the xylem from host root to host shoot, or from host to parasite, was calculated by multiplying the concentration of each element in the xylem sap at the base of the tobacco stem by the flux of water from host root to parasite or host root to host shoot over the experimental period. The flux of water was calculated from the amount of water lost in transpiration plus © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 937–947

the amount of water incorporated into tissues over the experimental period. The increment of each element in each plant part was calculated between 38 and 49 d after inoculation with O. cernua. To calculate the amount of each element supplied or removed by the phloem, the difference between that supplied by the xylem and the measured increment was calculated.

RESULTS Plant growth and carbon lost from respiration The increase in root dry weight of plants parasitized by O. cernua over the experimental period was lower than that of control plants (Table 1). The dry weight gain of O. cernua was equivalent to approximately 23% of the entire gain in the infected tobacco plant. Over the experimental period, the amount of carbon lost by respiration from shoots of infected and uninfected plants was similar, but the roots from infected plants lost less carbon through respiration than uninfected plants (Table 1). However, carbon lost by the parasite through respiration was greater than that of control roots.

Nutrient accumulation by the parasite and by the host In general, the relative elemental increments of the nutrients measured between the two harvests were similar in the roots and shoots of infected and uninfected plants (Table 1). There were, however, a number of notable exceptions. First, the amount of carbon accumulated by the roots of infected plants was about half that of uninfected plants, and the relative accumulation rate was also lower. Second, the roots of infected plants also had lower increments of N, Mg, Na and S over the experimental period. However, the relative elemental increments were typically similar to those of controls (Table 1). Third, the relative elemental increment in N of infected shoots was less than that of control shoots. Comparison between infected tobacco plants and O. cernua showed that increments in carbon and potassium by the parasite were larger than those of the infected roots, whereas the increments in magnesium, sodium, calcium, phosphorus and sulphur of the parasite were lower than those of the host roots.

Collection and contents of the xylem sap of the host and the parasite The pressure at which xylem sap started to flow from an incision made in the stem base of uninfected tobacco was slightly lower than in infected tobacco (Table 2). No xylem sap flowed from O. cernua when the root system was pressurized, even up to pressures of 0·61 MPa. However, when the shoot of the tobacco host was removed and the tobacco stem resealed in the pressure chamber, sap flowed from O. cernua at 0·25 MPa. The cross-sectional area of xylem vessels in O. cernua was 10-fold lower than xylem vessels in

940 J. M. Hibberd et al. Table 1. The effect of the parasite O. cernua on the dry weight (g), respiration (mmol C per 11 d) and the elemental content per organ (mmol) of its tobacco host. Data are shown as the initial dry weight and elemental composition, the increments over the 11 d, and the relative elemental increment (mmol mmol–1 d–1) and dry weight increment (g g–1 d–1) over the 11 d experimental period from 62 to 73 d after sowing. Tobacco plants were separated into the shoot and the root, the parasite was removed from the host roots. Data are shown as means ± SE; n = 4 to 8 Uninfected tobacco

Dry weight

Initial Increment Rel. increment

Respiration

Infected tobacco

Shoot

Root

Shoot

Root

O. cernua

6·39 ± 0·38 6·88 ± 0·66 0·066

1·72 ± 0·11 1·88 ± 0·20 0·067

5·93 ± 0·63 6·91 ± 0·89 0·070

1·34 ± 0·21 0·93 ± 0·15 0·048

1·95 ± 0·56 1·83 ± 0·97 0·060

22·67 ± 2·20

34·75 ± 3·74

28·25 ± 3·63

20·27 ± 3·31

55·22 ± 16·26

C


219·6 ± 14·9 227·6 ± 21·2 0·065

62·2 ± 4·2 58·8 ± 9·8 0·061

194·8 ± 21·3 205·2 ± 41·8 0·065

44·5 ± 5·9 26·6 ± 9·9 0·043

58·8 ± 17·1 79·5 ± 8·2 0·077

N


6·02 ± 0·51 3·01 ± 0·19 0·063

2·40 ± 0·14 1·67 ± 0·17 0·048

6·19 ± 0·72 2·96 ± 0·47 0·035

1·55 ± 0·25 1·14 ± 0·12 0·050

1·37 ± 0·32 1·63 ± 0·56 0·071

K


5·25 ± 0·38 2·30 ± 0·31 0·033

1·98 ± 0·29 0·30 ± 0·06 0·013

5·41 ± 0·59 2·42 ± 0·63 0·034

0·37 ± 0·06 0·27 ± 0·10 0·050

0·78 ± 0·17 0·53 ± 0·18 0·047

Mg


0·934 ± 0·050 0·744 ± 0·075 0·053

0·655 ± 0·080 0·297 ± 0·053 0·034

1·070 ± 0·091 0·917 ± 0·18 0·056

0·182 ± 0·043 0·155 ± 0·072 0·056

0·029 ± 0·005 0·020 ± 0·006 0·048

Na


0·147 ± 0·012 0·149 ± 0·021 0·064

0·578 ± 0·081 0·776 ± 0·148 0·077

0·234 ± 0·036 0·189 ± 0·049 0·054

0·271 ± 0·053 0·176 ± 0·081 0·046

0·039 ± 0·017 0·029 ± 0·021 0·050

Ca


2·204 ± 0·136 1·71 ± 0·188 0·052

0·515 ± 0·079 0·415 ± 0·088 0·054

1·955 ± 0·128 1·659 ± 0·244 0·056

0·216 ± 0·035 0·285 ± 0·101 0·077

0·019 ± 0·018 0·022 ± 0·026 0·070

P


0·771 ± 0·063 0·777 ± 0·188 0·063

0·570 ± 0·093 0·310 ± 0·101 0·040

0·729 ± 0·043 0·733 ± 0·123 0·063

0·177 ± 0·032 0·216 ± 0·083 0·073

0·109 ± 0·023 0·097 ± 0·034 0·058

S


0·673 ± 0·053 0·37 ± 0·052 0·040

0·359 ± 0·035 0·234 ± 0·36 0·046

0·769 ± 0·117 0·371 ± 0·101 0·036

0·110 ± 0·019 0·060 ± 0·026 0·040

0·048 ± 0·010 0·048 ± 0·017 0·063

the tobacco host (Table 2). The calculated pressure (using the Hagen-Poiseuille equation) needed for sap flow in the tobacco host was lower than the empirical pressure used to collect sap from tobacco.The calculated pressure needed for sap flow in O. cernua was the same as the pressure at which sap flowed experimentally. Typically, the concentrations of cations and anions in the sap of infected and uninfected plants were similar (Table 2). However, there was a large reduction in the concentration of PO4 in the xylem sap of infected tobacco plants, and malate was detectable in the xylem sap of control plants but not that of infected plants. The concentration of amino acids in the xylem sap of infected tobacco was only about 30% of that of uninfected control plants (Fig. 1a). The concentration of most amino acids in the xylem sap of O. cernua was higher than that collected from infected tobacco plants, however, the concentration of glutamine, GABA and methionine in the xylem

sap were lower in O. cernua than in infected tobacco (Fig. 1a & b). The activity of nitrate reductase in roots of plants infected with O. cernua was significantly lower than in control roots, and in O. cernua was extremely low when compared with the activity in tobacco (Table 2). Although the concentrations of almost all ions in the xylem sap of O. cernua were much higher than those in the xylem sap of uninfected or infected tobacco, the amino acid concentration of O. cernua xylem was only slightly higher. The increase in the concentration of all elements in the xylem sap of O. cernua can be related to the lower flow rate from O. cernua than from tobacco. The less pronounced increase in the amino acid concentration of xylem sap from O. cernua is likely to be due to lower rates of nitrate reduction in the roots of infected plants caused by both O. cernua and also removal of the host shoot during the treatment to collect xylem sap from O. cernua. Removing the host shoot © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 937–947

Withdrawal of tobacco nutrients by Orobanche 941 Table 2. The flux of water from root to shoot (ml d–1), the cross-sectional area of xylem vessels (mm2), the pressure (MPa) at which xylem sap flowed in tobacco and O. cernua, the calculated pressure (MPa) at which xylem sap would flow due to resistance from the xylem vessels, the in vivo activity of nitrate reductase (mmol NO2–g–1 fresh weight h–1), and the concentration (mM) of cations and anions in the xylem sap of tobacco and O. cernua at the root to shoot interface. Samples were taken from uninfected tobacco, infected tobacco and O. cernua. n.d. = not determined. n.det. = not detected. Balancing pressure in parentheses: measured after excising the host shoot and then resealing the root. Data are shown as means ± SE; n = 3 to 8

Water flux (ml d–1) Xylem vessel area (mm2) Balancing pressure (MPa) Calculated pressure (MPa) NR activity (mmol N02– g–1 fwt h–1) K Mg Na Ca Cl PO4 NO3 SO4 Malate

Uninfected tobacco

Infected tobacco

O. cernua

104·9 71·7 ± 2·7 0·38 ± 0·02 0·0002 1·52 ± 0·25 6·93 ± 0·71 0·89 ± 0·09 0·45 ± 0·08 1·57 ± 0·15 2·81 ± 0·41 0·67 ± 0·1 4·55 ± 0·85 0·54 ± 0·08 0·23 ± 0·05

132·0 n.d. 0·45 ± 0·02 n.d. 0·39 ± 0·09 4·80 ± 3·67 0·73 ± 0·10 0·39 ± 0·12 1·20 ± 0·52 1·87 ± 0·23 0·27 ± 0·05 4·17 ± 0·83 0·37 ± 0·07 n.det.

1·6 6·9 ± 0·5 n.d. (0·25) 0·25 0·11 ± 0·08 19·4 ± 3·67 1·78 ± 0·01 0·41 ± 0·02 1·85 ± 0·02 8·35 ± 3·29 4·4 ± 0·32 15·5 ± 1·23 2·69 ± 0·67 3·99 ± 1·37

would decrease the recycling of amino acids in the phloem, and also transfer from phloem to xylem. The concentration of calcium in the xylem sap of O. cernua was higher than in xylem sap of the tobacco host. However, the increase in calcium concentration in O. cernua xylem sap was very close to that predicted from the relationship between the concentration of calcium in the sap and the pressure used to collect that sap (data not shown).

Estimation of net flows within the infected host and to the parasite from the host Infection by O. cernua stimulated the amount of carbon fixed by the host plants by 20% when compared with their uninfected counterparts (Fig. 2a). In addition, a larger proportion of the fixed carbon was directed to the host roots

and the associated O. cernua, than was partitioned to the roots of control plants. However, the roots of infected plants received less carbon than those of the control plants, because O. cernua removed 73% of the carbon supplied to the roots of infected plants. Infection by O. cernua also reduced the amount of carbon being recycled to the shoots in the host xylem. The content of carbon in the xylem of tobacco infected by O. cernua could supply less than 1% of that which O. cernua accumulated over the experimental period. The increment in nitrogen of infected tobacco plants together with the parasite O. cernua was greater than that of uninfected plants (Fig. 2b). As the root system of infected plants was smaller than that of the control plants, the rate of uptake of nitrogen per unit weight of roots was stimulated 127% by parasitism. In both uninfected and infected

Figure 1. The concentration (mm) of the total amino acid pool and individual amino acids in the xylem sap of the parasitic plant O. cernua and also its infected tobacco host and uninfected controls. (a) The concentration of total amino acids, and abundant individual amino acids. (b) The concentration of less abundant amino acids. TOT, total amino acid pool; GLN, glutamine: ASN, asparagine; GAB, Gaba; GLU, glutamic acid; LYS, lysine; ASP, aspartate; THR, threonine; SER, serine; PRO, proline; GLY, glycine; ALA, alanine; VAL, valine; MET, methionine; CYS, cysteine; ILE, isoleucine; LEU, leucine; TYR, tyrosine; PHE, phenylalanine; ORN, ornithine; HIS, histidine; ARG, arginine. © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 937–947

942 J. M. Hibberd et al.

Figure 2. Empirical model of the net carbon (a) and nitrogen (b) flows in the xylem (black arrows) and in the phloem (dotted arrows) over an 11 d study period in uninfected tobacco and tobacco infected by the root parasitic angiosperm Orobanche cernua. The width of arrows (net flows via xylem and phloem), area of squares (C and N increments), and area of circles (C lost through respiration) are drawn in proportion to the rates of flows, increments or losses. Note that the scale of arrows and squares for nitrogen are 20-fold larger than for carbon.

tobacco there was a large cycling of N in the xylem and phloem. Roots of infected plants accumulated slightly less N than roots of control plants. The amount of N measured in the xylem sap of the infected tobacco could supply only 5% of the N that O. cernua accumulated over the study period (Fig. 2b), the amount in the xylem sap of the parasite would only supply 15% of the N it accumulated over the study period. Parasitism of tobacco by O. cernua did not influence the increment in K of shoot or root over the experimental

period (Fig. 3a). However, O. cernua accumulated more K than did the infected root to which it was attached, and as a result, the cycling of K in the xylem and phloem of the host was lower than in control plants. Accumulation of Ca in the roots of infected plants was lower than in roots of control plants, but O. cernua accumulated less than 10% of that which the root accumulated (Fig. 3b). More than 90% of the Ca that O. cernua received during the study period could be supplied from Ca in the host xylem. Infection reduced the increment in Mg of infected roots relative to © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 937–947

Withdrawal of tobacco nutrients by Orobanche 943

Figure 3. Empirical model of the net potassium (a) and calcium (b) flows in the xylem (black arrows) and in the phloem (dotted arrows) over an 11 d study period in uninfected tobacco and tobacco infected by the root parasitic angiosperm Orobanche cernua. The width of arrows (net flows via xylem and phloem), area of squares (K and Ca increments), are drawn in proportion to the rates of flows or increments. Note that the scale of arrows and squares for calcium are 2·5-fold larger than for potassium.

controls, however, 62% of the Mg that O. cernua accumulated could be supplied from the host xylem (Fig. 4a). The amount of Na accumulated by the roots of infected plants was lower than that accumulated by the roots of control plants. The amount of Na that O. cernua accumulated was 17% that of roots of infected plants; 22% of this sodium could be supplied from the host xylem (Fig. 4b). The roots of tobacco infected with O. cernua accumulated less S than control plants. This was due to withdrawal of S from both the xylem and phloem (Fig. 4c), however, 13% of S could be supplied from the host xylem. © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 937–947

DISCUSSION The pressure required for xylem sap to flow from an incision at the stem base of tobacco infected by O. cernua was slightly higher than that for uninfected tobacco. Balancing pressure is a function of the soil water potential and the rate of canopy transpiration (Passioura 1980). As our data showed that water lost through transpiration from infected plants was greater than that from uninfected plants, the higher balancing pressures for infected plants was probably due to their higher rates of canopy transpiration. The

944 J. M. Hibberd et al. greater loss of water from infected plants was probably due to delayed senescence of older leaves when compared with uninfected controls (Hibberd et al. 1998). We were unable to collect xylem sap from O. cernua while the host shoot was present. But, when the shoot of tobacco was removed (stopping the competing flux of water from host root to shoot in the transpiration-stream), sap did flow from O. cernua, and, at a lower pressure than for tobacco. From the

dimensions of the xylem vessels (using the HagenPoiseuille equation), we calculated the theoretical pressure that would be needed to move water through the xylem of the host and the parasite. Interestingly, the calculated conductance of the xylem elements within tobacco was much larger than the collective conductance we measured. These data indicate that the rate of transpiration is relatively more important that the xylem conductance in determining water

Figure 4. Empirical model of the net magnesium (a), sodium (b) and sulphur (c) flows in the xylem (black arrows) and in the phloem (dotted arrows) over an 11 d study period in uninfected tobacco and in tobacco infected by the root parasitic angiosperm Orobanche cernua. The width of arrows (net flows via xylem and phloem), area of squares (Mg, Na and S), are drawn in proportion to the rates of flows or increments. The model of the flows of sulphur is based on the concentration of sulphate in the xylem; the small concentration of sulphur-containing amino acids have not been considered. © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 937–947

Withdrawal of tobacco nutrients by Orobanche 945

Figure 4. Continued.

flux in tobacco, and, agrees well with data from a similar calculation for wheat (Passioura 1980). However, the calculated conductance of the xylem vessels of O. cernua and the overall conductance were remarkably similar, indicating that the conductance of parasite xylem vessels must be a large determinant of the conductance to water flow within its xylem.

Does O. cernua increase the flux of carbon towards the roots? We have previously shown that O. cernua led to an increase in dry matter partitioning to the host root and the parasite, compared with the roots of uninfected controls (Hibberd et al. 1998). The data presented here allow the first full quantification of carbon fluxes within a plant infected with a root holoparasite. The presence of O. cernua on the tobacco roots led to an increase (84%) in the net flux of carbon moving from the shoot to the root; however, 73% of this carbon was removed from the host roots by the parasite. Therefore, consistent with our first hypothesis, the presence of the parasitic sink not only resulted in a greater proportion of carbon moving from host shoot to host root, but also led to the diversion of host assimilate from the host root to the parasite. Over the experimental period, net fixation of C was 20% higher in tobacco infected by the root parasite O. cernua than in controls. This is similar to the situation reported for plants infected by the shoot parasite C. © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 937–947

reflexa (Jeschke & Hilpert 1997; Jeschke, Baig & Hilpert 1997). Interestingly, the root hemiparasite Olax phyllanthi and its host Acacia littorea showed the opposite response: the host fixed less carbon than did uninfected controls. In tobacco that is infected with O. cernua, the increase in the rate of carbon acquisition is probably due to delayed senescence of older leaves (Hibberd et al. 1998). In contrast, in associations involving the shoot parasite, C. reflexa, higher rates of photosynthesis occurred in young leaves as well as older ones (Jeschke & Hilpert 1997; Jeschke et al. 1997). Higher rates of net photosynthesis within the canopy induced by the different parasites may be demand-dependent. The fact that the rate of photosynthesis was increased in different regions of the canopy by the two parasites may be due to the relative proximity between leaves and the sites of parasite attachment.The mechanism/s by which partitioning of carbon within a plant is controlled is controversial. Minchin, Thorpe & Farrar (1993) proposed a simple mechanistic model based on the Münch hypothesis that was supported by experimental evidence. The model predicts that changes in the partitioning of carbon between two sinks can be driven by both an alteration in the kinetics of substrate uptake by one of the sinks, but also by the supply of substrate from the source leaf. It is therefore possible that the greater flux of carbon supplied to the host root by the shoot was due to both the ability of O. cernua to remove host carbon from the root and the increase in carbon fixed by the infected plants compared with controls.

946 J. M. Hibberd et al.

Does carbon supplied from the host to parasite originate from the host xylem? By measuring the amount of carbon in the host xylem as close as possible to the site of parasite attachment, and the flux of water into the parasite, we calculated that the host xylem could passively supply less than 1% of the carbon which O. cernua removed from the host. The concentration of C in the xylem sap of O. cernua would also only provide the parasite with less than 1% of the C that it accumulated over the study period. These data are consistent with our second hypothesis: that the xylem does not supply a significant proportion of the carbon removed by the parasite from the host. Thus, more than 99% of the carbon removed from the host by O. cernua is likely to have come from host phloem. The data are also interesting when compared with other studies in which attempts have been made to quantify the proportion of carbon supplied to parasitic plants by the host xylem stream. The shoot parasite Cuscuta reflexa, which has low rates of transpiration, removed less than 1% of its carbon from the host xylem (Jeschke et al. 1994b) a remarkably similar value to the one calculated for O. cernua. Carbon in the host xylem could supply 44% of the carbon that the hemiparasite Olax phyllanthi removed from its Acacia host (Tennakoon et al. 1997), although in this case it was not possible to include the amount of carbon lost through respiration in the analysis. In terms of the other solutes that are supplied to the parasite from the host xylem, the data indicate that the minimum values for the passive supply of each solute vary considerably, and, correspond to the concentration of each element in the host xylem sap. Our data also show a difference between the composition of host and parasite xylem sap, indicating that either the parasite selectively absorbs solutes from the host xylem, or that additional solutes move into the parasite xylem from the tubercle. In some fruits, excess water that enters the fruit via the phloem is removed via the xylem. We calculate that influx of water into the parasite via the phloem is lower than the total demand for water by the parasite (data not shown). However, we can not discount that, for limited time periods, excess water derived from the phloem moves back into the host via the xylem.

Does infection by O. cernua reduce the amount of carbon recycled in the xylem? Infection of tobacco by O. cernua reduced the amount of carbon moving from root to shoot in the xylem. As the amount of carbon moving from shoot to root in infected plants was stimulated, the proportion of carbon entering the root and then being recycled in the xylem was four-fold lower than that recycled in control plants. Less carbon moving from root to shoot in infected plants could not be accounted for by the amount that was supplied to the parasite in the host xylem. Lower concentrations of amino acids, probably due to lower activities of nitrate reductase in the roots of infected plants, contributed to the decreased

concentration of carbon in the xylem of infected plants compared with uninfected controls. We propose that the activity of nitrate reductase is lower in roots of infected plants because of the low supply of carbon to the roots down-regulating its activity (Glaab & Kaiser 1993) and also the expression and synthesis of protein (Vincentz et al. 1993). The presence of the shoot parasite C. reflexa led to lower amounts of amino acids in the root pressure xylem exudate of its R. communis and C. blumei hosts (Jeschke & Hilpert 1997; Jeschke et al. 1997). It was proposed that lower carbohydrate supply led to decreased activity and amount of nitrate reductase protein, although no direct evidence for this was given. In contrast, the root hemiparasite, O. phyllanthi, led to slightly higher rates of nitrogen fixation by nodules attached to the host Acacia littorea (Tennakoon et al. 1997). The absence of malate in the xylem of infected plants was also contributing to the lower amount of carbon recycled in the xylem of infected tobacco. Malate could be measured in significant quantities in the xylem of control plants. Interestingly, the concentration of malate from plants infected with C. reflexa, tended to be greater than in the control plants hosts (Jeschke & Hilpert 1997; Jeschke et al. 1997).

Does lower carbon supply to roots lead to lower rates of nutrient uptake and metabolism within the root? By calculating the increments of elements in host tissue and parasite tissue between the two harvests we have estimated the amount of each element taken up by the tobacco roots during the experimental period. In general, for the elements measured there was little difference in the rates of uptake over the experimental period between infected and uninfected tobacco plants. Exceptions were nitrogen, in which uptake was stimulated, and sodium, in which uptake was lower in infected plants compared with uninfected plants. It is surprising that the amount of nitrogen taken up by roots of infected plants was higher than uninfected plants. When expressed per unit weight of tobacco root, the rate of nitrate uptake was stimulated by O. cernua by 127%. In the association between A. littorea and the xylem-tapping root parasite O. phyllanthi, the rate of nitrogen uptake was similar in infected plants and controls (Tennakoon et al. 1997). However, increased rates of nitrate uptake have been reported in associations between the shoot parasite C. reflexa and various host species (Jeschke & Hilpert 1997; Jeschke et al. 1997). It was suggested that increased rates of uptake of N could be stimulated by (a) lower amino acid content within the root stimulating the uptake of N, and (b) higher rates of transpiration allowing increased flux across the rhizosphere (Jeschke & Hilpert 1997; Jeschke et al. 1997). An alternative is that the smaller root system of parasitized plants simply represents the portion of the root that is functional in the uptake of nitrogen. In which case, in uninfected plants much of the root system would not take up N. Therefore, although a lower supply of carbon to © 1999 Blackwell Science Ltd, Plant, Cell and Environment, 22, 937–947

Withdrawal of tobacco nutrients by Orobanche 947 infected roots coincided with both decreased root growth and nitrogen reduction, the rate of uptake of most ions was similar in infected and uninfected tobacco. In conclusion, our data indicate that the root holoparasite O. cernua is likely to rely almost exclusively on the host phloem for its supply of carbon and nitrogen. The phloem is also likely to be quantitatively more important for the supply of most of the other elements studied. Orobanche cernua influenced host nutrient fluxes in a similar manner to the shoot parasite C. reflexa. Both parasitic plants have a high dependence on host phloem for the supply of solutes. In contrast, O. phyllanthi was more dependent on the host xylem and differed in the way in which it influenced nutrient fluxes within the host (Tennakoon et al. 1997). Concerning interactions between plant parasites and their hosts in general, it would be interesting to determine if the rate of parasite transpiration could predict the degree to which that parasite removed solutes from the host xylem. Infection of tobacco by O. cernua represents an excellent system with which to investigate up-regulation of nitrate uptake and carbon fixation in a plant possessing an additional sink.

ACKNOWLEDGMENTS We thank Ralph Bungard (Sheffield) for help with the nitrate reductase assay, Andrea Hilpert, Marion Bernhard, Elfiede Reisberg and Eva Wirth (Würzburg) for technical assistance. We also thank John Pate (Nedlands) for valuable suggestions and insights. We acknowledge financial support from the BBSRC (50/PO1777), and the Sonderforschungsbereich 251 of the Deutsche Forschungsgemeinschaft.

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