Host-Species-Dependent Physiological Characteristics of Hemiparasite

Tree Physiology 34, 1006–1017 doi:10.1093/treephys/tpu073

Research paper

Host-species-dependent physiological characteristics of hemiparasite Santalum album in association with N2-fixing Downloaded from and non-N2-fixing hosts native to southern China

J.K. Lu1, D.P. Xu1, L.H. Kang1,4 and X.H. He2,3,4 http://treephys.oxfordjournals.org/ 1Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangdong 510520, China; 2Department of Environmental Sciences, University of Sydney, Eveleigh, NSW 2015, Australia; 3School of Plant Biology, University of Western Australia, WA 6009, Australia; 4Corresponding authors ([email protected], [email protected])

Received April 8, 2014; accepted July 25, 2014; published online September 12, 2014; handling Editor Heinz Rennenberg

Understanding the interactions between the hemiparasite Santalum album L. and its hosts has theoretical and practical significance in sandalwood plantations. In a pot study, we tested the effects of two non-N2-fixing Bischofia( polycarpa (Levl.) Airy

Shaw and Dracontomelon duperreranum Pierre) and two N2-fixing hosts (Acacia confusa Merr. and Dalbergia odorifera T. Chen) on the growth characteristics and nitrogen (N) nutrition of S. album. Biomass production of shoot, root and haustoria, N and

15 at South China Institute of Botany, CAS on October 8, 2014 total amino acid were significantly greater in S. album grown with the two N2-fixing hosts. Foliage and root δ N values of

S. album were significantly lower when grown with N2-fixing than with non-N2-fixing hosts. Significantly higher photosynthetic rates and ABA (abscisic acid) concentrations were seen in S. album grown with D. odorifera. Similarity in the proportional amounts of amino acid of root xylem sap between S. album and its host D. odorifera was also evident, suggesting major access to nitrogenous solutes from D. odorifera to S. album. Irrespective of host species, S. album clearly appeared to optimize xylem sap extraction from its hosts by higher transpiration and lower water-use efficiency than its host. The growth of two non-N2-fixing hosts parasitized by S. album was significantly greater than the equivalent values for unparasitized treatments, and lower growth and photosynthesis were observed for parasitized A. confusa, and significant decreases in root N, photosynthesis and transpiration for parasitized D. odorifera compared with unparasitized treatments. Furthermore, foliage ABA concentrations were significantly higher in all hosts parasitized by S. album than in their unparasitized counterparts. Our study is probably the first to report on host dependence and preference in the hemiparasite S. album, and the gener- ated results may have important implications for understanding of the physiological interactions between host species and parasitic plants, and for successfully mixing plantations of S. album with D. odorifera.

Keywords: abscisic acid, amino acids, δ15N, haustorium, net leaf photosynthesis rate, transpiration.

Introduction relying on their host plants to take up water and nutrients Among parasitic angiosperms, the sandalwood species through the haustorium, a specialized absorbing structure of (Santalum spp.), such as the most precious trees (S. album, parasitic plants, especially for nitrogenous compounds (Pate S. acuminatum and S. spicatum), parasitizes the roots of host 2001, Bell and Adams 2011). Hence, host preference is an plants (Tennakoon et al. 1997a, 1997b, Radomiljac et al. important aspect from which the parasite could gain measure- 1999c). Santalum species are hemiparasites as they have func- able benefit Jiang( et al. 2008, Irving and Cameron 2009, Bell tional chloroplasts to perform photosynthesis while partially and Adams 2011).

© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] Host-species-dependent physiological characteristics of Santalum album 1007

Indian sandalwood (S. album L.) has been over-exploited for photosynthesis and other physiological characteristics (Watling its aromatic heartwood and root, which have cosmetic, reli- and Press 2001, Cameron et al. 2005, 2008, Li et al. 2012, gious and medicinal significance Kim( et al. 2005, Ochi et al. Fisher et al. 2013). These effects could substantially influence 2005, Dhanya et al. 2010). Over the last two decades, several the growth and survival of host and/or parasitic plants. Thus, large-scale plantations of S. album have been established to studies on how interactions between hosts and parasites are meet future market requirements in Australia, China, India and important in selecting host plants for S. album plantation have Indonesia (Dhanya et al. 2010). Experiments on both pot and been carried out. field plantations have shown that the growth performance of We therefore investigated the host dependence and prefer- S. album is greatly enhanced by its successful attachment to ence in the growth and performance of the parasitic S. album. suitable hosts, particularly to N2-fixing species (e.g., Acacia, Four different host species (two N2-fixing legumes Acacia

Casuarina and Sesbania) (Radomiljac et al. 1999a, Li 2003). confusa Merr. and D. odorifera, and two non-N2-fixing plants Nevertheless, these hosts have a relatively low market value Bischofia polycarpa (Levl.) Airy Shaw and Dracontomelon dup- and the timely screening of suitable high-value host trees for erreranum Pierre) have been reported as superior host can- Downloaded from S. album plantations is needed. didates of S. album native to southern China (Li 2003). We Dalbergia odorifera T. Chen, one of most precious rose- aimed to address the following questions: (i) if S. album could woods in the world with diverse medicinal and commercial successfully parasitize host plants, what physiological altera-

values (Yu et al. 2007), has been successfully planted with tions did the host have after parasitization? (ii) Did the growth http://treephys.oxfordjournals.org/ S. album in the Jianfengling Arboretum of Hainan Island, China of S. album depend on its host species characteristics, e.g.,

(18°42′N, 108°49′E), since 1989. Recently, we have reported non-N2-fixing vs 2N -fixing host? If yes, among these four host an appropriated detailed understanding of N transfer between species which one could be the best candidate to provide a parasitized D. odorifera and S. album (Lu et al. 2013). This brand-new mixed plantation pattern of host and S. album? investigation has shown substantial N transfer from D. odorifera Answers to these questions could improve our understanding to S. album, while N2-fixation of D. odorifera could enhance of the physiological interactions between S. album and differ- the N transfer. Nevertheless, a further understanding of the ent host species in order to identify better mixed patterns for physiological interactions between S. album and D. odorifera is S. album field plantations in China. needed. As far as is known, no studies have investigated the at South China Institute of Botany, CAS on October 8, 2014 direct or indirect effect of a host on S. album parasitism, which Materials and methods may provide useful management strategies for D. odorifera/ S. album mixed plantations, and potentially provide significantly Plant material increased value to the existing sandalwood plantation industry Two N2-fixing legumes of A. confusa and D. odorifera and two with another high-value woody species. non-N2-fixing plants of B. polycarpa and D. duperreranum were Growth performance, nutrient elements, organic solutes, gas selected as hosts for S. album. Seeds of these five woody spe- exchange, haustorial anatomy and hormone variation have been cies were surface-sterilized with 3% sodium hypochlorite for studied between S. album and its respective hosts, including 5 min and rinsed with distilled water. Seeds of S. album were

N2-fixing hosts Acacia ampliceps, Acacia trachycarpa, Sesbania also soaked in 0.1% gibberellic acid (GA) for 12 h and rinsed formosa, and non-N2-fixing hosts Eucalyptus camaldulensis, with distilled water to promote germination (Radomiljac et al. Kuhnia rosmarinifolia and Tithonia diversifolia (Radomiljac et al. 1999a). The rinsed seeds were germinated on sterilized sand 1999a, 1999b, 1999c, Tennakoon and Cameron 2006, Zhang at 28–30 °C for ∼2–3 weeks until germination. Two emerging et al. 2012, Yang et al. 2014). Differences in nutrient concentra- seedlings with 1 cm radicals, either in a single species or with tion, amino acid concentration and composition of xylem saps, S. album as a pair, were moved into one 15 × 11 × 14 cm plastic photosynthesis and transpiration rates, δ15N and δ13C values, pot with 250 g potting mix (vermiculite : perlite = 2 : 1, v/v, pH when S. album is associated with different host species in these 6.8 ± 0.2) in April 2009. Seedlings were fertilized with 2.5 ml studies, have been used alongside parasite growth to explain of Hoagland solution once weekly and watered twice weekly why some hosts are markedly superior to others in terms of to field capacity. Seedlings of 2 N -fixing hosts were inoculated overall benefit to the parasite. Generally, legumes, rather than with Bradyrhizobium elkanii DG, an effective strain isolated non-legumes, are superior hosts due to their N2-fixation capac- from Pterocarpus macarocarpus Kurz. nodules (Lu et al. 2012). ity to provide the parasite with abundant N. However, some Meanwhile, seedlings of non-N2-fixing hosts were also inocu- contrary evidence demonstrated that host N is not always a lated with the same amounts of autoclaved B. elkanii inoculants. reliable predictor of parasite performance (Atsatt and Strong 1970, Kelly 1990, Marvier 1996, Jiang et al. 2008). Experimental design On the other hand, parasitic plants have deleterious In April 2010, parasitic associations were established direct and indirect effects on their host in terms of growth, between 1-year-old S. album (20 ± 1.2 cm height and

Tree Physiology Online at http://www.treephys.oxfordjournals.org 1008 Lu et al.

3.3 ± 0.3 mm diameters) seedlings and their four 1-year-old Root xylem sap was collected by an aspirator (A-1000S; host species. One parasitized pair was then transplanted to a EYELA, Tokyo, Japan, Figure 1) from freshly excavated root 40 × 30 × 5 3 cm pot containing 5 kg of the above-mentioned branches. To collect root sap, root segments were tightly potting mix. A 20-cm distance was fixed between these two inserted into a plastic pipette tip and a mild vacuum (−0.04 seedlings in each pot to reduce distance effects. In addition, to −0.07 MPa) was then applied to the apparatus via a rubber two individuals of each species were also transplanted into one hose that was connected to an aspirator (Pate et al. 1994). pot for determination of intraspecific competition. As a result, Approximately 300 µl xylem sap for each treatment was then with three replicates or pots for each paired association plants, bulked and frozen for further analyses. a total of 72 pots were grown in a greenhouse in a random Numbers of haustoria of S. album on each host plant were arrangement under 33/25 °C day/night and 65/75% day/night counted and harvested from those forming typical appressed relative humidity, at the Research Institute of Tropical Forestry contacts with root xylem and those directly parasitizing root in Guangzhou (23°11′N , 113°23 ′E), China. Plants were watered nodules on N2-fixing hosts. three times weekly to near field capacity and fertilized with Downloaded from 15 50 ml of Hoagland nutrition monthly. Analyses of N and N natural abundance Total N in plant foliage and roots was determined by the Measurements of leaf photosynthesis and transpiration Kjeldahl digestion method (Knowles and Blackburn 1993). δ15N

Using a LI-6400 portable photosynthesis system with a stan- values of leaves and roots were analyzed using an isotope ratio http://treephys.oxfordjournals.org/ dard 6-cm2 leaf chamber (LI-COR, Inc., Lincoln, NE, USA), net mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, −2 −1 rates of leaf photosynthesis (Pn, µmol CO2 m s ) and transpi- MA, USA) at the State Key Laboratory of Tree Genetics and −2 −1 ration (E, mmol H2O m s ) were measured 6 months after Breeding in Beijing, China. transplantation between 09:30 and 10:30 am on two consecu- tive sunny days in October 2010. Photosynthetic irradiance Abscisic acid analysis was provided by an integrated red-blue light-emitting diode The second, third and fourth fully expanded leaves of both source (Model 6400-02B; LI-COR, Inc.) at 1000 µmol m−2 s−1. S. album and hosts were selected to measure their abscisic acid

During measurements on these 2 days, the CO2 concen- (ABA) concentrations. Briefly, 0.5 g deep frozen tissue, col- tration inside the greenhouse was 387.8 ± 4.7 ppm. Leaf lected from a small strip in the middle portion of these leaves, at South China Institute of Botany, CAS on October 8, 2014 temperature was 30 °C. To ensure a similar leaf age and devel- but avoiding their mid-rib, was ground with 2 ml of 80% metha- opment, the third, fourth and fifth fully expanded phyllodes of nol containing butylated hydroxy toluene (0.001%, w/v). The A. confusa and leaves of S. album, and the second, third and extract was mechanically shaken (120 rpm) for 1 h at 4 °C and fourth fully expanded leaves of B. polycarpa, D. duperreranum then subjected to centrifugation at 13,000g for 20 min. The and D. odorifera were sampled. Water-use efficiency (WUE, supernatants were assayed for ABA using an enzyme-linked −1 µmol CO2 mmol H2O ) as Pn/E was then calculated. immunosorbent assay with the monoclonal antibody (AFRC MAC 252; Sigma, Poole, Dorset, UK) (Asch et al. 2001). Plant harvesting and sampling Six months after transplantation, the shoot and root of S. album Analyses and identifications of amino acids and its host plants were separately harvested on 25 October The collected root xylem sap (300 µl) was left at room tem- 2010, oven dried at 70 °C for 72 h and then ground with a ball mill perature, and then diluted 10 times with 3% trichloroacetic (Retsch GmbH & Co. KG, Haan 1, Germany) for further analyses. acid solution and centrifuged at 10,000 rpm for 15 min. A total

Figure 1. Conceptual diagram of xylem sap collection. AS, aspirator; EP, Eppendorf tube; PG, pressure gauge; PT, pipette tip; RH, rubber hose; S, rubber stopper; SF, suction flask.

Tree Physiology Volume 34, 2014 Host-species-dependent physiological characteristics of Santalum album 1009 of 20 µl collected supernatant was injected into an L-8800 ; ,x ,x ,y ,y α γ α High Speed Amino Acid Analyser (HITACHI High-Technologies, β 2.70 1.20 0.32 Tokyo, Japan). Separation was primarily achieved on a HITACHI 0.24

± ± ± ±

column 855-350, and the reaction temperature was 134 °C. 2.10 The analyses of amino acids were followed with the instrumen- 5.79 14.01 S. album 10.78 tal manual using the ninhydrin reaction methods. The identifi- followed the by 3) = n ), or in rows between), a cations of amino acids were compared with the standard amino γ , SE, SE,

β ± , acid solutions, type AN and type B, which were purchased α 3.24a,x 3.23a,x 1.88b,x 2.54b,x , Dracontomelon duperreranum

from the Wako Pure Chemical Industries, Osaka, Japan. ± ± ± ±

9.14

Statistical analysis 20.74 30.63 22.35 Host plant Root g/plant DW Data (means ± SE, n = 3) were subjected to one-way analysis Downloaded from ,x ,x ,y ,y γ α β α of variance and the significant differences between treatments ; Dracontomelon were compared using the least significant difference at the 4.71 1.96 0.88 6.86

± ± ± ±

P = 0.05 level. The impact of S. album on hosts was assessed

by comparing the parasitized and unparasitized hosts using 7.02 17.61 28.71 32.80 S. album

Student's t-test. All analyses were performed with SPSS ver- http://treephys.oxfordjournals.org/ sion 11.5 (SPSS Inc., Chicago, IL, USA). grown with four different hosts ( , Dalbergiaodorifera 3.67a,x 3.24a,x 3.82a,x 3.93b,y S. album

± ± ± ±

Results

Growth performances ; Dalbergia 31.35 Host plant 32.63 19.49 35.21 Shoot DW g/plant Shoot DW In general, significantly greater diameter, height, shoot and root ,y ,x ,y ,y β α β biomass of S. album were observed in seedlings associated α grown in pots under greenhouse conditions. Values (means with the two N -fixing hosts A. confusa and D. odorifera than 1.08 0.49

2 0.50 0.34

at South China Institute of Botany, CAS on October 8, 2014 ± ± ± ±

in seedlings with the two non-N2-fixing hosts B. polycarpa and and between c) b, (a, S. album , Bischofia polycarpa

D. duperreranum (Table 1). 5.15 5.47 8.17 8.70 S. album

Height, diameter and total biomass of both non-N2-fixing S. album hosts parasitized by S. album were significantly greater than the equivalent values for the unparasitized treatment (Student's ; Bischofia 0.60a,x 0.85b,x 0.98c,x 0.86b,x

± ± ± ±

t-test; P < 0.05) (see Table S1 available as Supplementary Data at Tree Physiology Online). In contrast, lower growth indices 7.04 11.93 Diameter mm/plant 14.24 11.96 were observed for parasitized A. confusa but not for D. odorifera Host plant

compared with the unparasitized treatment (see Table S1 avail- confusa , Acacia ,x ,x ,x α ,x α β able as Supplementary Data at Tree Physiology Online). β 13.3 5.7 3.9 10.8

± ± ± ±

Haustorial number and biomass production 82.3 84.3 After 1 year of growth with host plants, haustorium had been 0.05.< Acacia 121.4 136.4 S. album successfully formed between roots of S. album and its host (data not shown). Bell-shaped S. album haustoria attached to

the roots of both 1.5-year-old N2-fixingA. confusa and D. odor- at P y) (x, 2.0b,y 22.7a,x 4.0b,y 1.9b,y

± ± ± ±

ifera (Figure 2a and b) and non-N2-fixing B. polycarpa and D. duperreranum (Figure 2e and f). Meanwhile, S. album haus- 57.0 57.8 69.0 S. album

104.5 Host plant Height cm/plant Height toria also penetrated into 1.5-year-old N2-fixing A. confusa

and D. odorifera nodules (Figure 2c and d). All haustoria were dry. DW, weight. classified into three size categories:< 0.2 cm, 0.2–0.5 cm and -fixing plants 2

>0.5 cm. The majority of haustoria were <0.2 cm in most spe- Santalum /

cies, and there were significantly more haustoria of this size -fixing plants 2 Santalum Santalum / formed on B. polycarpa than on other host species (Table 2). / Santalum Santalum album , Santalum G /

Numbers of 0.2–0.5 cm haustoria were significantly higher 1 in D. odorifera than in other host species (Table 2). In addi- Bischofia Dracontomelon Dalbergia Acacia Grown with non-N sameletter are not significantly different in columns between host species grown with hostspecies and its associated Table . rowthcharacteristics four 1.5-year-old of plant hosts and their paired hemiparasite Grown with N tion, D. odorifera had significantly greater biomass production Santalum association Plant

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Figure 2. Haustoria of 1.5-year-old hemiparasite S. album attached to host roots and nodules. (a) Root of A. confusa, (b) root of D. odorifera, (c) nodule of A. confusa, (d) nodule of D. odorifera, (e) root of B. polycarpa and (f) root of D. duperreranum. (a and d) Scale bar = 0.5 mm; (b, c and e) scale bar = 2 mm; (f) scale bar = 1 mm. AcN, A. confusa nodule; AcR, A. confusa root; BpR, B. polycarpa root; DoN, D. odorifera nodule; DoR, D. odorifera root; DdR, D. duperreranum root; Hstr, haustorium; SaR, S. album root.

Table. 2 Number of hemiparasite S. album haustoria attached to host roots and N2-fixing nodules grown in pots under greenhouse conditions. Values (means ± SE, n = 3) followed by the same letter are not significantly different in columns between host species grown with S. album (a, b, c) or in rows between numbers of different sized haustoria on roots (x, y, z) at P < 0.05. at South China Institute of Botany, CAS on October 8, 2014 Host species Number of haustoria on roots Number of Total attached Total biomass of Biomass per growth status haustoria on haustoria attached haustoria attached >0.5 cm 0.2–0.5 cm <0.2 cm nodules mg/plant haustorium mg

Grown with non-N2-fixation plants B. polycarpa 2.0 ± 1.0b,z 9.0 ± 3.0b,y 70.0 ± 5.0a,x 79.7 ± 2.1a 102.3 ± 21.6c 1.2 ± 0.3c D. duperreranum 5.0 ± 1.0a,z 13.0 ± 4.0b,y 35.0 ± 10.0b,x 57.0 ± 5.6b 122.3 ± 29.6b 2.1 ± 0.4b

Grown with N2-fixation plants A. confusa 3.0 ± 1.0b,y 9.0 ± 5.0b,x 15.0 ± 7.0c,x 5.0 ± 3.0a 37.7 ± 6.1c 116 .0 ± 20.5c 3.2 ± 0.5a D. odorifera 4.0 ± 1.0a,y 21.0 ± 2.0a,x 26.0 ± 7.0b,x 6.0 ± 3.0a 58.0 ± 12.3b 200.0 ± 14.4a 3.7 ± 0.2a

of attached haustoria than other host species (Table 2). significantly higher in S. album when associated with N2-fixing Moreover, the biomass of per haustorium was generally signifi- hosts than with non-fixing hosts (Figure 3). Foliage N con- cantly higher in N2-fixing hosts than in non-fixing hosts, except centrations were significantly higher in S. album than in their when comparing A. confusa with D. duperreranum (Table 2). non-N2-fixing hosts, whereas root N concentrations were lower 2 Numbers of haustoria (r = 0.29, P = 0.07, n = 12) were in S. album than in their N2-fixing hosts. In addition, S. album not correlated with S. album biomass production, while bio- seedlings associated with D. duperreranum showed consider- mass production of all haustoria (r2 = 0.44, P = 0.02 n = 12) ably lower N concentrations in both foliage and roots than in and per haustorium (r2 = 0.79, P = 0.00, n = 12) in the hosts seedlings associated with other hosts. Amounts of plant total significantly correlated with biomass production in S. album N were significantly higher in 2 N -fixing than in non-N2-fixing (see Figure S1a and b available as Supplementary Data at plants, while they were without significant differences in plant

Tree Physiology Online). In addition, comparatively low num- total N between N2-fixing or between non-N2-fixing species bers of haustoria penetrated nodules in the two N2-fixing hosts (Figure 3c). A. confusa and D. odorifera (Figure 2c and d; Table 2). Root N concentrations of D. odorifera were significantly lower in parasitized than in unparasitized treatments, but N concen- Nitrogen concentrations trations in foliage and root were significantly higher in parasit-

Concentrations of both foliage and root N and total amounts ized than in unparasitized non-N2-fixing hosts (see Table S1 of plant (all aboveground and belowground tissues) N were available as Supplementary Data at Tree Physiology Online).

Tree Physiology Volume 34, 2014 Host-species-dependent physiological characteristics of Santalum album 1011

Foliage and root δ15N values Foliage and root δ15N values were significantly higher in non-

N2-fixing hostsB. polycarpa and D. duperreranum (4.56–4.62 and

3.21–3.99‰) than in N2-fixing hostsA. confusa and D. odorifera (0.31–0.43 and 1.18–2.01‰) (Figure 4). Foliage and root δ15N values of S. album were significantly lower when associated with

N2-fixing hosts −( 0.23 to 1.22‰ and 1.29–1.64‰) than with

non-N2-fixing hosts (2.33–3.62 and 2.40–2.93‰) (Figure 4).

Amino acid profiles in xylem sap Total concentrations of amino acids in the xylem sap were gen-

erally higher in the N2-fixing hosts than in the non-N2-fixing hosts (Figure 5). This was also true for S. album when it was Downloaded from

associated with an N2-fixing host. The principal amino acid in the host root xylem var- ied between species (Figure 5). Aspartic acid dominated in

A. confusa, hydroxylysine in B. polycarpa and phosphoserine http://treephys.oxfordjournals.org/ at South China Institute of Botany, CAS on October 8, 2014

Figure 3. Concentrations of nitrogen (N) in foliage (a) and roots (b) and total (aboveground + belowground) amounts of plant N (c) in Figure 4. Foliar and root δ15N values of 1.5-year-old paired hemiparasite 1.5-year-old paired hemiparasite S. album (white bars) and its four plant S. album (open squares) and its four plant hosts (filled squares). Three rep- hosts (black bars). Data are means ± SE (n = 3) and bars denoted by licates were performed for each treatment. For each plant tissue, data are different letters are significantly different P( < 0.05) between four par- means ± SE (n = 3) and bars denoted by different letters are significantly asitized host species grown with S. album (a, b, c), between S. album different (P < 0.05) between four parasitized host species grown with grown with different hosts (α, β, γ), and between host and hemipara- S. album (a, b, c), between S. album grown with different hosts (α, β, γ, δ), site for each pair (x, y). Acacia/Santalum, A. confusa with S. album; and between host and hemiparasite for each pair (x, y). Acacia:Santalum, Bischofia/Santalum, B. polycarpa with S. album; Dalbergia/Santalum, A. confusa with S. album; Bischofia:Santalum, B. polycarpa with S. album; D. odorifera with S. album; Dracontomelon/Santalum, D. duperreranum Dalbergia:Santalum, D. odorifera with S. album; Dracontomelon:Santalum, with S. album. D. duperreranum with S. album.

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Figure. 5 Percentage of amino acid composition (molar basis of the total amino acids) in the root sap of 1.5-year-old paired hemiparasite S. album and its four plant hosts. Acacia/Santalum, A. confusa with S. album; Bischofia/Santalum, B. polycarpa with S. album; Dalbergia/Santalum, D. odorif- at South China Institute of Botany, CAS on October 8, 2014 era with S. album; Dracontomelon/Santalum, D. duperreranum with S. album. Ala: alanine; Ans, anserine; Asp, aspartic acid; Cys, cystine; Cysthi, cystathionine; Glu, glutamic acid; Gly, glycine; Hylys, hydroxylysine; Ile, isoleucine; Leu, leucine; Met, methionine; PEA, phosphorylethanolamine; Pro, proline; P-Ser, phosphoserine; Sar, sarcosine; Ser, serine; Val, valine; Thr, threonine; others include β-aminobutyric acid, γ-aminobutyric acid, histidine, hydroxyproline, lysine, ornithine, phenylalanine and tyrosine. Mean total amino acid concentrations (nmol l−1) and numbers of amino acid types are given in parentheses above bars.

in other plant species, i.e., the N2-fixing D. odorifera and non- S. album associated with either non-N2-fixing or 2N -fixing hosts

N2-fixing D. duperreranum. Phosphoserine was the most com- was significantly higher than its corresponding hosts P( < 0.05), mon amino acid in the xylem sap of S. album when associated except A. confusa (Figure 6a). In addition, foliage ABA was sig- with any host. Other major amino acids in the xylem sap were nificantly higher in all hosts when parasitized by S. album than similar between S. album grown with N2-fixing hosts and its their unparasitized counterparts (P < 0.05) (see Table S2 avail- hosts. However, the amino acid composition of root xylem sap able as Supplementary Data at Tree Physiology Online). in S. album differed from that of their associated non-N2- fixing Amounts of foliage ABA were significantly higher in hosts, B. polycarpa and D. duperreranum. For instance, both N2-fixing hosts A. confusa and D. odorifera (10.48 and parasitized non-N2- fixing hosts contained aspartic acid and 11.00 ng per plant) than in non-N2-fixing hosts B. polycarpa glutamic acid, whereas glutamine or asparagine was absent and D. duperreranum (7.57 and 7.27 ng per plant) (Figure 6b). in S. album associated with B. polycarpa or D. duperreranum . Santalum album L. grown with N2-fixing hosts had significantly higher total foliage ABA (13.38 and 20.75 ng per plant) Concentrations and amounts of ABA than with non-N2-fixing hosts (1.13 and 1.29 ng per plant) Acacia confusa had significantly higher foliar ABA concentra- (Figure 6b). Interestingly, foliage ABA concentrations were tion than other hosts, whether they were in the parasitized or higher but foliage ABA amounts were lower than their hosts unparasitized treatments (Figure 6a, see Table S2 available as when S. album grew with the two non-N2-fixing hosts. Supplementary Data at Tree Physiology Online). Foliage ABA was highest in S. album grown with D. odorifera (0.62 ng g−1 Pn, Tr and WUE for different host–parasite pairings DW), followed by with A. confusa (0.52 ng g−1 DW), then with There were no clear trends between the photosynthesis rates −1 D. duperreranum (0.43 ng g DW) and least with B. polycarpa (Pn) of S. album and its hosts (Figure 7). Values of Pn were (0.26 ng g−1 DW) (Figure 6a). The ABA concentration of significantly higher in parasitizedA. confusa and B. polycarpa,

Tree Physiology Volume 34, 2014 Host-species-dependent physiological characteristics of Santalum album 1013 Downloaded from http://treephys.oxfordjournals.org/ at South China Institute of Botany, CAS on October 8, 2014

Figure 6. Concentrations (a) and amounts (b) of foliage ABA in 1.5-year-old paired hemiparasite S. album (white bars) and its four plant hosts (black bars). Data are means ± SE (n = 3) and bars denoted by different letters are significantly different P( < 0.05) between four parasitized host species grown with S. album (a, b, c), between S. album grown with different hosts (α, β, γ), and between host and hemiparasite for each pair (x, y). Acacia:Santalum, A. confusa with S. album; Bischofia:Santalum, B. polycarpa with S. album; Dalbergia:Santalum, D. odorifera with S. album; Dracontomelon:Santalum, D. duperreranum with S. album. but significantly lower in parasitized D. duperreranum and D. odorifera, than their corresponding parasite S. album. Figure. 7 Photosynthesis, transpiration and water-use efficiency of Values of P in S. album were enhanced by N -fixing hosts 1.5-year-old paired hemiparasite S. album and its four plant hosts. n 2 Data are means ± SE (n = 3) and bars denoted by different letters are −2 −1 (5.96 and 8.23 µmol CO2 m s in A. confusa and D. odor- significantly different P( < 0.05) between four parasitized host species ifera) but decreased by non-N2-fixing hosts (3.69 and grown with S. album (a, b, c), between S. album grown with different −2 −1 hosts (α, β, γ), and between host and hemiparasite for each pair (x, y). 4.49 µmol CO2 m s in B. polycarpa and D. duperreranum) Acacia:Santalum, A. confusa with S. album; Bischofia:Santalum, B. poly- (Figure 7). Values of Pn were significantly higher in non- carpa with S. album; Dalbergia:Santalum, D. odorifera with S. album; N2- fixing hosts B. polycarpa and D. duperreranum, but sig- Dracontomelon:Santalum, D. duperreranum with S. album. nificantly lower in 2N -fixing hosts A. confusa and D. odorifera, under parasitized than unparasitized conditions (P < 0.05, In all associations, transpiration rates (Tr) were significantly see Table S2 available as Supplementary Data at Tree higher in S. album than in its associated hosts (Figure 7). Rates

Physiology Online). of Tr were similar between the parasitized and unparasitized

Tree Physiology Online at http://www.treephys.oxfordjournals.org 1014 Lu et al.

non-N2-fixing hosts, but were significantly higher or lower because some attached haustoria are not able to penetrate in parasitized N2-fixing A. confusa or D. odorifera than in their vascular tissues (Cameron et al. 2006, Cameron and Seel their unparasitized counterparts (see Table S2 available as 2007, Suetsugu et al. 2012). Supplementary Data at Tree Physiology Online). Values of WUE were significantly higher in any associated Nitrogenous solute flux from host to S. album host plant than in S. album (Figure 7). The WUE values Since the xylem sap of host and parasite are linked by a haus- D. duperreranum were significantly higher in parasitizedA. confusa torium, it has been suggested that organic N is acquired by and D. duperreranum, but similar in parasitized B. polycarpa and the passive, non-selective flux of xylem sap from the host to D. odorifera, than in their corresponding unparasitized counter- the parasite (Pate et al. 1994, Radomiljac et al. 1998, Hibberd parts (see Table S2 available as Supplementary Data at Tree and Jeschke 2001, Pageau et al. 2003). Our results revealed Physiology Online). substantial and consistent differences between the amino acid composition of the root xylem sap in the host species and associated S. album (Figure 5). The concentrations of amino Downloaded from Discussion compounds in the root xylem sap of the two N2-fixing hosts

This study investigated a number of interrelated physiological and associated S. album were higher than the two non-N2- fixing factors involved in the complex interactions between S. album hosts and their associated S. album (Figure 5). Therefore, our

and its four potential plant hosts native to China, growing in results provide evidence that a direct bulk transfer of major http://treephys.oxfordjournals.org/ pairs in pots under greenhouse conditions, with a view towards nitrogenous solute did indeed take place from the xylem of developing a reliable D. odorifera/S. album mixture plantation in N2-fixing hosts to S. album. This finding is consistent with the southern China. ‘nitrogen parasitism hypothesis’ that the primary physiological function of importing organic N compounds from the host Growth responses on S. album xylem sap is to supply N to the parasite (Schulze et al. 1984,

The two N2-fixing species A.( confusa and D. odorifera) used in Marshall et al. 1994). However, a total of 13 or 19 amino acids this study were better hosts than the two non-N2-fixing hosts with two distinctive organic acids (pipecolic acid and djenkolic (B. polycarpa and D. duperreranum) in promoting S. album acid) were detected (Tennakoon et al. 1997b, Radomiljac et al. growth (Table 1). This is in accordance with previous stud- 1998), while a total of 26 amino acids were found in this study at South China Institute of Botany, CAS on October 8, 2014 ies, which concluded that xylem-tapping root hemiparasites (see Figure 5). We assume these differences are mainly from grow better when associated with N2-fixing hosts, apparently the utilization of different plants species and tissues (stem sap as a result of higher N concentrations in the xylem of legumes vs root sap). Meanwhile, a new generation of amino acids ana- compared with non-legumes (Radomiljac et al. 1999b, 1999c, lyzer was also employed in this study. Press and Phoenix 2005, Bell and Adams 2011). The data for The abundant nodulation of A. confusa and D. odorifera, a the two N2-fixing plants in the present study clearly showed number of haustoria of S. album attached to both nodules and that D. odorifera was better at promoting the growth and roots, and values of δ15N close to zero for both legumes and photosynthesis of S. album than A. confusa, but was consid- accompanying S. album provide evidence of a high depen- erably poorer than the latter species when considering foliar dence of host and parasite on fixed N. For most annual or N. Nevertheless, by the end of the 6-month study period, the woody hemiparasites, the proportion of nitrogenous compound growth of A. confusa was severely constrained under parasit- transferred from the host ranged widely, from low (0.2–18%) ism. Thus, our results indicate that S. album possibly parasitizes via non-N2-fixing hosts to high (56–70%) via 2N -fixing plants various angiosperm hosts, but some are more heavily infected (Tennakoon et al. 1997b, Jiang et al. 2004, Cameron and Seel than others. 2007); in such studies there is little relevance in distinguish-

An interesting finding from this study was that the growth ing between N2-fixation and N transfer. Bearing this in mind, of S. album in the absence of a host was greater than that the present study examines that D. odorifera with effective of plants grown with non-N2-fixing host B. polycarpa and nodules do have enhanced N fluxes between host D. odorif-

D. duperreranum (data not shown), supporting earlier studies era and S. album, suggesting that effective N2-fixation could suggesting a minimal benefit, if not disadvantage, in terms of supply more nitrogenous substances in the xylem sap to the competition for nutrients when the parasite associates with an S. album haustoria (Lu et al. 2013). Hence, N2-fixing legumes inferior host (Radomiljac et al. 1999a). Our studies also found are assumed to be superior hosts by supplying N to parasites. that the most suitable host quality index for S. album were the In contrast, some studies have reported that N2-fixing species biomass production per haustoria and the total haustorial bio- do not enhance the growth of parasites compared with non- mass production instead of the number of haustoria attached N2-fixing hosts Atsatt( and Strong 1970, Kelly 1990, Marvier to the host's roots. This is in accordance with previous studies 1996, Matthies 1997). Recently, Jiang et al. (2008) suggested that not all species attacked by parasites act as suitable hosts that N2-fixation does not influence the quality of legumes as

Tree Physiology Volume 34, 2014 Host-species-dependent physiological characteristics of Santalum album 1015 hosts for the hemiparasite Rhinanthus minor, but rather the Thus, we would intend to investigate whether such an effect well-developed haustorium formed by the parasite, coupled is responsible for accelerating the heartwood formation of with the lack of defensive mechanism of the host, and the D. odorifera after parasitization by S. album. presence of ample nitrogenous compounds in the xylem sap accessible to the parasite's haustorium, govern the host qual- Gas exchange between S. album and host ity of legumes. Our results show that the comparison of δ15N The results of this study showed that increased N derived from the signatures of the parasite and its potential hosts gives a good xylem stream of the two N2-fixing hosts resulted in an increase of indication of the N2-fixing plants likely to act as principal N N concentration in S. album tissue and high photosynthetic per- sources to S. album. formance and ultimately, improved biomass production (Table 1 and Figure 3). Improved rates of photosynthesis in S. album when ABA concentrations in parasite and host grown in association with N2-fixing A. confusa and D. odorifera Parasitic plants are known to accumulate high concentrations of provide circumstantial evidence that S. album has a lower depen- Downloaded from ABA (Jiang et al. 2010), but its function in such unprecedented dence on such hosts for carbon compared with non-N2-fixing concentrations remains unclear. In any case, parasitization may B. polycarpa and D. duperreranum. However, S. album clearly significantly influence the associated host metabolism to favor appears to optimize root xylem sap extraction from its hosts in nutrient acquisition by the parasite, such as elevated levels the same way as the obligate hemiparasite Striga hermonthica

of ABA in host tissues (Taylor et al. 1996, Frost et al. 1997, (Taylor et al. 1996) and facultative hemiparasite Rhinanthus minor http://treephys.oxfordjournals.org/ Pageau et al. 2003). In common with other hemiparasites, such (Jiang et al. 2003), by having higher transpiration rates and lower as Striga and Rhinanthus species, the foliar ABA concentration WUE values than in the host. However, notable exceptions include in S. album was found to be significantly higher than in its asso- S. acuminatum (Tennakoon et al. 1997a), Olax phyllanthis (Pate ciated hosts (Figure 6). In addition, the foliar ABA concentration et al. 1990) and mistletoe (Press et al. 1993, Seel et al. 1993). in all four hosts was significantly higher after attachment by Due to elevated parasite transpiration, xylem sap is drawn S. album, as shown earlier for Striga and Rhinanthus by Taylor through the haustorium, providing vascular continuity between et al. (1996) and Frost et al. (1997). Taylor et al. (1996) sug- Striga and its host, and into the parasite via cohesion (Press and gested three possible sources for the extra ABA following host/ Graves 1995). But there is no vascular continuity between the parasite association: (i) a wounding response by haustorial haustorium of S. album and its host Tithonia diversifolia, instead at South China Institute of Botany, CAS on October 8, 2014 attachment; (ii) a water deficiency of the host after parasitizing; the presence of massive interfacial parenchyma was found at the (iii) ABA synthesis in the parasite and transport into the host. host–parasite interface (Tennakoon and Cameron 2006). A recent study found another mechanism by which haustoria of S. album could synthesize phytohormones (e.g., ABA and GA), Host responses to parasitism which was likely to be used for cell division and differentiation It has long been known that parasitism can suppress the bio- during haustorial development (Zhang et al. 2012). mass and photosynthesis of the associated host plant (Hibberd In this study, a strong positive relationship was observed et al. 1996, Watling and Press 2001, Cameron et al. 2005, between S. album biomass and amounts of ABA (r2 = 0.83, 2008, Fisher et al. 2013). In Dalbergia, significant decreases in P = 0.00, n = 12, see Figure SIC available as Supplementary photosynthesis, transpiration and N concentration of root were Data at Tree Physiology Online), suggesting that ABA con- noted compared with unparasitized plants (see Table S2 avail- tent might be a reliable indicator of host quality. Zhang et al. able as Supplementary Data at Tree Physiology Online), suggest- (2012) verified that the ABA and GA contents of S. album ing that S. album sequesters significant amounts of N from the were three times higher in attached haustoria than in non- host plant. These decreases were mirrored in Acacia, except for attached haustoria, suggesting that endogenous hormones the N concentration and transpiration (see Table S2 available as (e.g., indole-3-acetic acid, GA and ABA) might be involved in Supplementary Data at Tree Physiology Online). In contrast, in the haustorial formation of S. album and in water and nutri- the two non-N2-fixing plants B. polycarpa and D. duperreranum, ent transport for the host–parasite association (Zhang et al. intraspecific competition had a worse influence on the growth of 2012). Based on the haustorial research, our explanation for unparasitized plants than parasitism by S. album (see Table S1 the high concentration and amounts of ABA in S. album grown available as Supplementary Data at Tree Physiology Online). From with superior hosts is that it is possible that S. album may an evolutionary perspective, in order to acquire large amounts of form more effective haustoria with suitable hosts (A. confusa xylem sap from hosts, parasites may have evolved by improving and D. odorifera) than with inferior hosts (B. polycarpa and their attraction to potential hosts rather than by their absorbing D. duperreranum) (Figure 6). Several studies have indicated ability. The hemiparasite S. album competed poorly with hosts that hormones (e.g., ABA, jasmonic acid) could be respon- B. polycarpa and D. duperreranum for available nutrients in pot sible for initiating the heartwood transformation (Davison studies, and thus both non-N2-fixing hosts parasitized byS. album and Young 1973, Shain and Hillis 1973, Taylor et al. 2002). grew consistently better than their unparasitized treatments (see

Tree Physiology Online at http://www.treephys.oxfordjournals.org 1016 Lu et al.

Table S1 available as Supplementary Data at Tree Physiology Atsatt PR, Strong DR (1970) The population biology of annual grass- Online). However, differences in defensive ability have been land hemiparasites. I. The host environment. Evolution 24:278–291. Bell TL, Adams MA (2011) Attack on all fronts: functional relationships indicated to underpin these differences in host quality and the between aerial and root parasitic plants and their woody hosts and associated parasite-induced host damage (Cameron et al. 2006, consequences for ecosystems. Tree Physiol 31:3–15. Cameron and Seel 2007, Rümer et al. 2007). Because of the Cameron DD, Seel WE (2007) Functional anatomy of haustoria formed absence of obviously defensive structures, Fabaceae have always by Rhinanthus minor: linking evidence from histology and isotope tracing. New Phytol 174:412–419. served as good hosts for R. minor (Rümer et al. 2007, Jiang et al. Cameron DD, Hwangbo JK, Keith A, Geniez JM, Kraushaar D, Rowntree 2008). Nevertheless, poor host Plantago lanceolata exhibited J, Seel WE (2005) Interactions between the hemiparasitic angio- strong reactions (e.g., releasing toxic secondary compounds and sperm Rhinanthus minor and its hosts: from the cell to the ecosys- host cell disintegration) against the haustorial tissues (Rümer tem. Folia Geobot 40:217–229. Cameron DD, Coats AM, Seel WE (2006) Differential resistance et al. 2007). Therefore, in investigations addressing S. album– among host and non-host species underlies the variable success of host interactions, anatomical surveys on host defense may be the hemi-parasitic plant Rhinanthus minor. Ann Bot 98:1289–1299. Downloaded from important to get a complete picture of the complex interactions. Cameron DD, Geniez JM, Seel WE, Irving LJ (2008) Suppression of In conclusion, the results on the physiology of the root hemi- host photosynthesis by the parasitic plant Rhinanthus minor. Ann Bot 101:573–578. parasite S. album presented in this study collectively provide Davison RM, Young H (1973) Abscisic-acid content of xylem sap. an insight into the complex interactions between the parasite Planta 109:95–98.

and the host during the early growth stage of S. album. A range Dhanya B, Syam V, Seema P (2010) Sandal (Santalum album L.) con- http://treephys.oxfordjournals.org/ of questions remain concerning the heartwood development servation in southern India: a review of policies and their impacts. of both S. album and its suitable host D. odorifera. Further J Trop Agric 48:1–10. Fisher JP, Phoenix GK, Childs DZ, Press MC, Smith SW, Pilkington research in this area is needed to allow the development of MG, Cameron DD (2013) Parasitic plant litter input: a novel indi- a superior methodology for the concurrent plantation of these rect mechanism influencing plant community structure. New Phytol two valuable timber species. 198:222–231. Frost DL, Gurney AL, Press MC, Scholes JD (1997) Striga hermonthica reduces photosynthesis in sorghum: the importance of stomatal limi- Supplementary data tations and a potential role for ABA? Plant Cell Environ 20:483–492. Hibberd JM, Jeschke WD (2001) Solute flux into parasitic plants. J Exp Supplementary data are available at Tree Physiology Online. Bot 52:2043–2049. at South China Institute of Botany, CAS on October 8, 2014 Hibberd JM, Quick WP, Press MC, Scholes JD (1996) The influence of the parasitic angiosperm Striga gesnerioides on the growth and Acknowledgments photosynthesis of its host, Vigna unguiculata. J Exp Bot 47:507–512. Irving LJ, Cameron DD (2009) You are what you eat: interactions The authors are indebted to Professor Bernard Dell of Murdoch between root parasitic plants and their hosts. Adv Bot Res 50: University and Dr Longbin Huang of University of Queensland, 87–138. Jiang F, Jeschke WD, Hartung W (2003) Water flows in the para- Australia for their valuable suggestions for experimental design. sitic association Rhinanthus minor/Hordeum vulgare. J Exp Bot 54: They also thank two anonymous reviewers and the handling 1985–1993. Editor Dr Heinz Rennenberg for their valuable comments on Jiang F, Jeschke WD, Hartung W (2004) Abscisic acid (ABA) flows the manuscript. from Hordeum vulgare to the hemiparasite Rhinanthus minor and the influence of infection on host and parasite abscisic acid relations. J Exp Bot 55:2323–2329. Conflict of interest Jiang F, Jeschke WD, Hartung W, Cameron DD (2008) Does legume nitrogen fixation underpin host quality for the hemiparasitic plant? None declared. J Exp Bot 59:917–925. Jiang F, Jeschke WD, Hartung W, Cameron DD (2010) Interactions between Rhinanthus minor and its hosts: a review of water, min- Funding eral nutrient and hormone flows and exchanges in the hemiparasitic association. Folia Geobot 45:369–385. This study was supported by the National Science Foundation Kelly CK (1990) Plant foraging: a marginal value model and coiling of China (31300527) and the Fundamental Research response in Cuscuta subinclusa. Ecology 71:1916–1925. Kim TH, Ito H, Hayashi K, Hasegawa T, Machiguchi T, Yoshida T (2005) Funds for the Central Non-profit Research Institution of CAF Aromatic constituents from the heartwood of Santalum album L. (CAFYBB2014QB011). Chem Pharm Bull 53:641–644. Knowles R, Blackburn TH (1993) Nitrogen isotope techniques. Academic Press, San Diego, CA. References Li AR, Smith FA, Smith SE, Guan KY (2012) Two sympatric root hemiparasitic Pedicularis species differ in host dependency and selectiv- Asch F, Andersen MN, Jensen CR, Mogensen VO (2001) Ovary ity under phosphorus limitation. Funct Plant Biol 39:784–794. abscisic acid concentration does not induce kernel abortion in field- Li YL (2003) Sandalwood introduction and research (in Chinese). grown maize subjected to drought. Euro J Agron 15:119–129. Science Press, Beijing, China.

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