Journal of Experimental Botany, Vol. 48, No. 310, pp. 1061-1069, May 1997
Journal of
Experimental
Botany
Xylem fluxes of fixed N through nodules of the legume
Acacia littorea and haustoria of an associated
N-dependent root hemiparasite Olax phyllanthi
Kushan U. Tennakoon1 and John S. Pate2
Department of Botany, The University of Western Australia, Nedlands 6907, Australia
Received 28 August 1996; Accepted 16 January 1997
Abstract
Nodulated 1-1.5-year-old plants of Acacia littorea
grown in minus nitrogen culture were each partnered
with a single seedling of the root hemiparasite Olax
phyllanthi. Partitioning of fixed N between plant organs
of the host and parasite was studied for the period 4 - 8
months after introducing the parasite. N fluxes through
nodules of Acacia and xylem-tapping haustoria of Olax
were compared using measured xylem flows of fixed N
and anatomical information for the two organs. N 2 fixation during the study interval (635 fig N g FW nodules 1
d 1) corresponded to a xylem loading flux of 0.20 //g
N mm" 2 d~ 1 across the secretory membranes of the
pericycle parenchyma of the nodule vascular strands.
A much higher flux of N (4891 fig mm 2 d~1) exited
through xylem at the junction of nodule and root. The
corresponding flux of N from host xylem across
absorptive membranes of the endophyte parenchyma
of Olax haustorium was 1.15 fig N m m " 2 d 1, six times
the loading flux in nodules. The exit flux from haustorium to parasite rootlet was 20.0 fig N m m " 2 d ~ \
200-fold less than that passing through xylem elements
of the nodule. Fluxes of individual amino compounds
in xylem of nodule and haustorium were assessed on
a molar and N basis. N flux values are related to data
for transpiration and partitioning of C and N of the
association recorded in a companion paper.
Key words: Olax phyllanthi, host-parasite relationships, N
flux, Acacia, N2 fixation.
Introduction
Studies of the nitrogen relationships between xylemtapping root hemiparasites and their hosts have so far
1
2
been restricted to a relatively few taxa, notably herbaceous
genera of the family Scrophulariaceae, such as Odontites
(Govier et al, 1967), Rhinanthus (Seel et al, 1993; Seel
and Press, 1993), Bartsia and Parentucellia (Press et al.,
1993), and Striga (McNally et al, 1983; Parker, 1984;
McNally and Stewart, 1987; Cechin and Press, 1993a, b,
1994; Press, 1995) and, among woody species, to the
shrub Olax phyllanthi of the Olacaceae (Pate et al, 1994).
Striga spp., by far the most intensively yet investigated,
occur mostly as weeds of monocultured cereals and
legumes, where parasites are attached to a single host
species, at least under agricultural conditions (Musselman
and Press, 1995). However, the rest of the above parasites
normally exploit a wide range of species in their respective
native habitats, thus rendering difficult evaluations of the
nature and extent of benefit derived from any one host.
This characteristic, combined with the possibility of the
parasite absorbing N from the soil independently of its
hosts (Press and Whittaker, 1993), contrasts noticeably
with the much simpler situation found in above-ground
parasites such as mistletoes (Pate et al, 1991a) and
dodders (Jeschke et al, 1994a, b) and their respective
hosts.
This programme of study on the N relationships of
Olax phyllanthi first examined the biology (Pate et al,
1990c), water relations (Pate et al, 1990a) and the xylem
inputs of amino acids from hosts to parasites (Pate et al,
1994) in native habitat. Then, using Olax partnered singly
in pot culture with identified major host species, growth
and N benefit to the parasite from certain hosts were
shown to outweigh greatly that from others (Tennakoon
and Pate, 1996a). One shrub legume, Acacia littorea,
turned out to be by far the most suitable of the hosts
studied and, when effectively nodulated and partnered
with Olax in minus N pot culture, proved capable of
Present address: Department of Botany, University of Peradeniya, Peradenrya, Sri-Lanka.
To whom correspondence should be addressed. Fax: +61 09 380 1001.
6 Oxford University Press 1997
1062
Tennakoon and Pate
meeting the full requirements of the parasite for N. At
the same time this host was demonstrated to provide
Olax with a heterotrophic input of carbon equivalent to
approximately one-half of the total increment of dry
matter carbon currently recorded for the parasite
(Tennakoon and Pate, 1996a).
Using the same system of symbiotically-dependent
Acacia parasitized by Olax, Tennakoon et al. (1997) then
examined the effects of parasitism on growth and resource
partitioning of the host and developed empirically-based
models depicting the respective inputs, exchanges and
utilizations of C and N by shoots, roots, nodules, and
haustoria of the associated species. That investigation
serves as a database for the present anatomically-based
study in which xylem loading and mean flux characteristics for N within the association are quantified between
nodules and receptor host plus parasite and across the
haustorial interface from host to parasite.
Materials and methods
Plant material
The study utilized pot grown sand cultures of 1-1.5-year-old
plants of Acacia littorea Masln., each plant partnered with a
single seedling plant of the root hemiparasite Olax phyllanlhi
(Labill) R. Br. The pots (4.5 1) received a nutrient solution
lacking nitrogen, but containing balanced amounts of all other
nutrient elements throughout the culture period. Harvesting,
processing and analysis of plant material of the two harvests
involved in all flux measurements were identical to that
described in the companion study of C and N partitioning
within the association (Tennakoon et al., 1997). The two
harvests were 122 d apart and took place at approximately 4
(early June 1995) and 8 months (early October 1995) after
transplanting the Olax seedling into the pots of already well
established Acacia in February 1995.
Measurement of mean flux of fixed N from nodules
A group of 25 randomly-selected nodules, encompassing a full
range of sizes and ages encountered on the Acacia plants of the
two harvests were weighed individually and their nonhaemoglobin-pigmented apical (meristematic) and proximal
(senescent) portions removed. The remaining part of the nodule
cylinder was presumed to be representing the site of N2 fixation.
This part of tissue was fixed whole in 2.5% glutaraldehyde in
0.025 M phosphate buffer (pH 7.0) and dehydrated and embedded in glycol methacrylate as described by O'Brien and Me
Cully (1981). Transverse sections (3.5-4.5 ^m thick) were then
taken at a series of levels throughout each nodule, stained with
0.05% toluidine blue (pH 4.2) and these used to assess mean
numbers of strands running longitudinally alongside the haemoglobin-pigmented region of each nodule (Plate 1A). The length
of the vascular strands lining the haemoglobin pigmented
bacterial tissue of a nodule was assessed as the mean functional
length involved in export of fixed N. The lengths of these
vascular strands were measured by mounting halved nodule
cylinders flat in water between microscope slides and examining
by means of a dissecting microscope and a micrometer eyepiece.
These measurements were combined with data for vascular
strand number to estimate combined mean functional length of
vascular tissue serving regions of the nodule involved in export
of fixed N. Then, knowing mean fresh weight of nodules, the
'working length' of vascular strand per gram of nodule
was assessed.
Assessments of mean flux of fixed N from bacterial tissue to
xylem of the nodule were made essentially following the
procedures and assumptions described earlier by Gunning et al.
(1974) for nodules of Pisum sativum. Loading of the nodule
xylem was assumed to proceed exclusively by selective release
of amino compounds from the cytoplasm of the pericycle cells
surrounding the conducting tissues of the nodule. This secretory
activity is presumed to enrich with amino compounds the
apoplastic compartment within the endodermis-invested vascular strands and thereby motivate osmotic attraction of water
into this vasculature (Pate and Gunning, 1972). Fixation
products are then pictured as being flushed out through the
nodule xylem elements into the main xylem stream of the root.
Unlike the situation in nodules of Pisum (Pate et al., 1969),
the pericycle cells of the vascular tissue of nodules of Acacia
littorea are not modified into transfer cells with wall ingrowths.
The surface of pericycle cells can therefore be measured as the
potential secretory surface within transverse sections of nodule
strands simply by using appropriately magnified light micrographs (Plate IB). The photomicrographs were scanned in
terms of combined surface area of their pericycle cells (PC,
Plate IB), using a Power Macintosh 7100/66 computer and the
public domain NIH image analysis programme developed at
the US National Institute of Health and available from the
internet by anonymous FTP from zippy.nimh.nih.gov.
Using an equivalent series of sections taken at the proximal
ends of the 25 selected nodules, assessments were also made of
the combined transectional area of lumina of xylem elements
present in vascular tissue at the junction between nodule and
parent root.
Assessments of mean N export from nodules to the
Olax-Acacia association was derived by:
NA =
[N,/(DxNFW)]
where iV, is the N increment in Acacia (minus nodules) plus
Olax over the study interval, D is the length of the study period
(d) and AVw the mean FW of nodules per plant over the
study interval.
The secretory fluxes of N across the pericycle cells of the
nodule vasculature or exiting through the xylem of the nodule
into the parent root were assessed by combining the appropriate
area-based anatomical assessments with measured rates of
fixation (7VA) during the study interval.
Measurement of mean flux of N from host to parasite via root
haustoria
A set of 25 randomly-selected mature haustoria of Olax, each
attached to a root of its Acacia host was used for determining
the area of endophytic tissue facing the exposed xylem of the
host. The endophytic junction of the haustorium with host
xylem is ellipsoidal in outline with the long axis oriented parallel
to the host root and with the concave absorptive face appressed
tightly against the exposed surface of the host stele (Pate et al.,
19906). The extent of curvature of this surface in transectional
view was assessed from low power light micrographs of a series
of transverse sections of parasitized host roots fixed and
embedded as described above for nodules (Plate 1C, D). Based
on such observations, a scaled macro replica formed from
modelling clay was constructed conforming precisely in relative
terms to the outer dimensions of the endophyte and the inner
curvature of its interfaces as demonstrated from the photographs. This scale model indicated that the values obtained for
Olax-Acacia N fluxes
1063
Plate 1. (A) Light micrograph of transverse section of the proximal end of nodule of Acacia littorea (close to point of attachment to rootlet)
showing peripherally-located vascular bundles [VB] in nodule cortex [C] surrounding bacterial tissue [B]. (B) Detailed anatomy of a transverse
section through a vascular bundle from the mid region of a nodule of Acacia littorea showing the loading parenchyma of the pericycle [PC] with
its cells (dotted) encircling the conducting elements of xylem [X] and phloem [P]. An endodermis [E] with well developed casparian thickenings
surrounds the pericycle parenchyma; cortex [C], (C) Transverse sections of mid region of a haustorium [H] of 0. phyllanthi. The endophytic tissue
[E] of the haustonum has penetrated the cortex of the host (A. littorea) to establish direct contact with the host stele [HS]. (D) Detailed anatomy
of the haustorial interface between O. phyllanthi and A. littorea. Demarcation between parasite and host root xylem tissue [HX] is marked by
arrows. Interface parenchyma of the parasite [P] show unevenly thickened walls. Note absence of xylem-to-xylem luminal contact between host
and parasite. (E) Transverse view of a haustorium sectioned close to the junction with the parent rootlet, showing exiting tracheary elements [X]
surrounded by several layers of cortical cells [C].
1064
Tennakoon and Pate
lengths of long and short axes of the endophyte would need to
be multiplied by a correction factor of 1.05 to convert the data
into actual surface area available for absorption from the host
xylem. Knowing mean fresh weight of haustoria, absorptive
area of endophyte per unit fresh weight of haustoria was then
determined.
Parallel measurements of xylem luminal area available for
the exit of N from the haustorium at its junction with the
parasite root were made using light micrographs of the proximal
ends of 25 haustoria sectioned transversely as close as possible
to their parent rootlets and stained with 0.05% toluidine blue
(Plate IE). Xylem lumen areas were analysed using the
computer-based image analysis system described above for
nodules, and, knowing weights of haustoria studied, data were
then expressed per mm2 of xylem lumen transectional area in
unit fresh weight of haustoria.
Assessments of mean N flux through haustoria assumed the
recorded increments of N in Olax (minus haustoria) between
the two harvests had been derived exclusively by haustorial
uptake of fixed N from the Acacia (Tennakoon and Pate,
1996a). Mean daily N fluxes per unit area (mm2) of endophyte
absorptive interface or per unit area (mm2) of xylem lumina
exiting at the haustorial-root junction were then expressed on
a g FW basis, i.e. in a manner directly comparable with that
outlined above for assessing mean N fluxes through nodules.
Assessment of export fluxes of individual amino compounds
through nodules and haustoria
Samples of xylem tracheal sap of nodule-bearing roots of the
Acacia and haustoria-bearing roots of Olax were collected from
each paired association at each harvest using the mild vacuum
mini extraction technique recently described by Pate et al.
(1994). Amino compounds were assayed in pooled sap samples
of host and parasite using the HPLC-based techniques described
by Pate et al. (1985) and Tennakoon and Pate (19966). Using
data for increment of plant N (minus nodules) in host plus
parasite over the study interval and amino acid composition of
Acacia root xylem sap, fluxes of each compound per unit weight
of nodules per day were then estimated on a molar or N basis.
Corresponding assessments were also made of amino acid fluxes
through haustoria, using values for increment of N in Olax
(minus haustoria) and amino acid composition data for tracheal
sap of Olax roots.
Results
Export mean fluxes of fixed N from nodules of Acacia
Table 1 gives data for dry weights, N concentrations in
dry matter and N contents of plants of Acacia (minus
nodules) and Olax (whole plants) for the two harvests.
Corresponding data are included for mean fresh weights
of nodules per plant for the respective harvests. These
data, derived from Tennakoon et al. (1997) are then used
to calculate the combined increment offixedN in parasite
plus host (minus nodules) in the study period and thereby
estimate the daily mean flux of fixed N through unit fresh
weight of nodules for the 122 d study interval. The
resulting value of 635 fig N g FW nodules"1 d"1
(Table 1), when related to an estimated secretory surface
of 3209 mm2 for the pericycle parenchyma surface of a
gram of nodules, translates to a mean xylem loading flux
across the membranes of these parenchyma of 0.20 fig
N mm" 2 d"1. Using the same procedure, but in this case
incorporating the mean transectional area (0.13 mm2) of
xylem lumina at the nodule:root junction of a gram of
nodules, the corresponding mean exit flux from the nodule
turns out to be a much greater value at 4891 fig
Nmm^d"1.
Fluxes of individual amino compounds through nodules (Table 2) were estimated on the assumption that the
nitrogenous solutes exporting fixed N were in the proportions indicated from analyses of tracheal sap of nodulebearing Acacia rootlets. Two major solutes, pipecolic acid
and asparagine, carried the major portion of the transported N and accordingly showed highest fluxes on a N
or molar basis. Djenkolic acid, aspartic acid and glutamic
acid were next in order of importance followed by a range
of minor compounds. The high levels of unusual nonprotein amino compounds (pipecolate and djenkolate)
suggest that their synthesis might occur in the nodule
through secondary metabolism of primary products (e.g.
from the glutamate, aspartate glutamine and asparagine)
formed in N2 fixation. Pipecolate and djenkolate feature
prominently in transport and storage of N in a number
of other Australian Acacia spp. (Pate et al., 1991a) and
have also been recorded as major solutes of xylem tracheal
sap of A. littorea in native habitats or pot culture (Pate
et al., 1994; Tennakoon and Pate, 1996a).
Mean fluxes of N through haustoria from Acacia to Olax
Table 3 provides data and procedures for estimating mean
flux of xylem-mobile fixed N through the haustoria of
Olax. The analysis combines information on N increment
of Olax (minus haustoria) for the study interval with
values for haustorial fresh weight per plant to derive a
mean flux through the haustoria of 279 fig N g FW" 1
d^.Then, using the estimated value of 244 mm2 g
haustoria"1 of endophyte surface against xylem of the
host, the above mean flux can be re-estimated in terms
of interface area to give a value of 1.15 fig N mm " 2 d "'.
Finally, incorporating the estimate of 13.9 mm2 of xylem
lumina area present at the junction between haustoria
and rootlet of the parasite, an estimated mean exit flux
of 20.0 ^g Nmm~ 2 d"1 is obtainedbetween haustorium
and parent Olax.
Fluxes of amino acids leaving the haustorium for the
parasite were estimated as above for nodules by proportioning the mean flux of total N through the haustorium
against individual amino compounds on the basis of their
respective molar or N concentrations in tracheal sap of
rootlets of the parasite. The resulting data (Table 4)
showed generally lower fluxes for all amino compounds
per unit weight of haustoria than was shown above for
nodules (cf. Tables 2, 4). Furthermore, xylem sap of Olax
was much more heavily biased towards asparagine than
Olax-Acacia N fluxes
1065
Table 1. Xylem export of fixed N from nodules to the parent plant of Acacia littorea and an attached root hemiparasite Olax
phyllanthi
Acacia-Olax association
Olax
(whole plant)
Acacia
(minus nodules)
Mean dry weight (g plant 1Y
N concentration in dry matter (%)
N content of dry matter (mg N plant"1)
Mean fresh weight of nodules (g plant"1)
First
harvest
Second
harvest
First
harvest
Second
harvest
33.8±2.1
0.99
335±21
1.53 + 0.19
44.8 ±2.4
0.92
409±22
3.68 + 0.43
2.85±0.50
1.10
31.5±5.3
15.0±1.7
0.96
143±17
Study period (d)
N increment in Acacia (minus
nodules) plus Olax over the study
interval (mg N)
Mean fresh weight of nodules per plant
over the study interval (gplant"1)
Mean N flux to the association (Olax +
Acacia) (jigNg FW nodule"1 d" 1 )
Mean length of vascular traces facing
haemoglobin-pigmented regions of
nodules (mm nodule"1)*
Mean number of vascular traces visible
in a transverse section of a nodule*
Mean circumference of loading
pericycle parenchyma*
(mm vascular trace"1)
Mean fresh weight per nodule (mg)'
Mean surface area of loading pericycle
parenchyma for combined functional
length of vascular trace
(mm2 g FW" 1 nodules)
Mean N loading flux into nodule
xylem across secretory surfaces of
pencycle parenchyma ((igNmm" 1 d"1)
Mean transectional area of lumina of
xylem elements exiting from nodule
into the rootlet (mm2g FW"1 nodules)
Mean N flux exiting to the association
through xylem elements at the
nodule:root junction
d"')
122
186±22
2.59±0.26
635 ±79
7.00±0.22
13±1
0.60 ±0.02
17.0± 16
3209 ±335
0.20 ±0.03
0.13±0.01
4891 ±613
"Total of 15 plants sampled and weighed at each harvest.
'Measurements taken on 25randomly-selectednodules harvested from the different Acacia plants of the two harvests.
that of Acacia and the novel amino compound S-ethenyl
cysteine, found so far only in Olax (Thumfort et ai,
1993; Pate et ai, 1994; Tennakoon and Pate, 1996a),
emerged as second in importance to asparagine in terms
of xylem transport of N. Its prevalence in rootlet sap
suggested possible synthesis in the haustorium from
incoming fixed N.
Discussion
A primary objective of this study was to assess the
effectiveness of xylem loading and export of N from the
nodules of a legume host and compare the mean fluxes
involved with comparable mean fluxes for host-derived
N through the xylem-tapping haustoria of an attached
root hemiparasite. Comparisons based on unit mass of
the organs concerned are of course confounded by the
fact that, nodules synthesize the fixed N which they are
exporting through their peripheral vasculature, whereas
the haustorium is primarily involved in absorbing and
processing N abstracted from the host xylem and reconstituting a xylem stream of N compounds which it then
exports to the parasite. These differences aside, membrane-mediated transfer of N leading to xylem export
from either organ can still be directly ascribed to specific
apoplast:symplast junctions. The operational interface of
the nodule is assumed to comprise the pericycle parenchyma which release amino compounds to the internal
apoplast of the vascular traces which surround the N2fixing bacterial tissue. In the haustorium the comparable
activity would involve absorption of amino compounds
from the apoplast of the xylem of the host by the interface
1066
Tennakoon and Pate
Table 2. Export fluxes for major amino compounds through nodules of Acacia littorea parasitized by Olax phyllanthi
Amino
compound
Pipecolic acid
Asparagine
Djenkolic acid
Aspartic acid
Glutamic acid
Senne
Glutamine
y-amino butyric acid
Alanine
Phenylalanine
Glycine
Others
Total
Estimated rate of flux through nodules
Xylem (tracheal) sap concentrations
(nodule-bearing Acacia rootlets)
of Acacia"
(^mol ml" 1 )
(^molgFW-'d"1)
UNgFW-'d"1)
16.9
8.02
2.59
2.43
1.40
0.85
0.49
0.49
0.49
0.36
0.32
1.76
36.2
237
224
36.3
34.0
19.6
11.9
13.9
6.9
6.8
5.05
4.52
33.3
635
2.71
1.28
0.42
0.39
0.23
0.14
0.08
0.08
0.08
0.06
0.05
0.28
5.80
(MgNmT 1 )
37.9
35.9
581
5.44
3.15
1.91
2.23
1.10
1.10
0.81
0.72
5.34
102
"Calculations based on mean rate of export of total N from nodules over the 122 d study interval (see Table 1) and the assumption that this flux
was proportioned on the basis of relative concentrations (N or molar basis) of amino compounds present in tracheal sap collected from nodulebearing rootlets of Acacia obtained from the two harvests
Table 3. Xylem export of host-derived N from haustoria to Olax phyllanthi when parasitizing the Nrfixing host A. littorea
Olax phyllanthi
(minus haustona)
Mean dry weight (g plant ')"
N concentration in dry matter (%)
N content of dry matter (mg N plant" 1 )
Mean fresh weight of haustoria (g plant" 1 )
Study period (d)
N increment in Olax (minus haustoria)
over the study interval (mg N plant" 1 )
Mean fresh weight of haustoria per
plant over the period of study (g plant" [ )
Mean N flux from host to Olax
U g N g FW haustoria" 1 d" 1 )
Mean fresh weight per haustorium (mg)
Mean area of endophytic interface
against host xylem (corrected for
curvature of interface)
(rrun2g FW haustoria "M"
Mean N flux into Olax per unit area of
endophytic interface (/ig N mm" 2 d" 1 )
Mean transectional area of xylem
elements exiting from haustorium into
the rootlet (mm 2 g FW haustoria" 1 )'
Mean N flux to Olax exiting through
xylem elements at the haustoria: parasite
root junction (^g N mm ~2 d ~')
First harvest
Second harvest
2.78 ±0.47
1.10
30.6 ±5.2
0.48±0.11
14.0±1.7
0.96
134± 16
5.29±0.41
122
104±17
2.80±0.21
279 ±34
3.27±0.30
244 ±16
1.15±0.14
13.9±2.6
20.0 ±2.4
"Total of 15 plants sampled and weighed at each harvest. Bulked samples were assyed for total N.
* Measurements taken on 25 randomly selected haustoria harvested for different Olax plants of the two harvests.
parenchyma of the endophyte of the parasite.
Furthermore, since both nodule and haustorium have
clearly prescribed vascular junctions with their subtending
roots, a further strictly comparable set of mean flux
measurements can be computed based on mean flux of N
via xylem lumina at these respective exit points to the
parent plants in question.
Viewed simply in terms of throughput on an organ
fresh weight basis, mean N flux from nodules, estimated
at 635 ^g N g FW" 1 d"1,turns out to be over twice that
through haustoria (279 /ug N g FW" 1 d" 1 ). This apparent
superiority in nodule functioning on a unit weight basis
applies despite upwards of 60-70% of the nodule's volume
being devoted to bacterial tissue involved in synthesis of
the exported solutes. However, on the other hand it must
be remembered that a substantial proportional volume of
Olax-Acacia N fluxes
1067
Table 4. Export fluxes for major amino compounds through haustoria to the parasite Olax phyllanthi
Amino
compound
Xylem (tracheal) sap concentrations
(haustoria bearing Olax rootlets)
Estimated rate of flux through haustoria of OIax°
ml ')
Asparagine
S-ethenyl
Cysteine
Proline
Aspartic acid
Senne
Glutamic acid
Glutamine
Pipecolic acid
Arginine
Glycine
Djenkolic acid
Others
Total
2.51
70.3
6.16
0.56
0.44
0.34
0.22
0.19
0.17
0.13
0.12
0.09
0.03
0.39
5.25
7.94
6.19
4.83
3.10
2.74
4.96
1.88
3.59
1.33
0.49
5.96
113
1.39
1.08
0.84
0.54
0.48
0.43
0.33
0.31
0.23
0.08
0.97
12.9
172
19.5
15.2
11.8
7.62
6.75
12.2
4.63
8.84
3.27
1.20
14.65
279
"Calculations based on mean rate of export of fixed N from haustoria over the 122 d study interval (see Table 3) and the assumption that this
flux was proportioned on the basis of relative concentrations (N or molar basis) of amino compounds present in tracheal sap collected from
haustoria-bearing rootlets of Olax obtained from the two harvests.
the haustorium is devoted to non-absorbing external
tissues, mostly comprising the appressorial disc attaching
the organ to a host root. Additionally, absorptive capacities of the endophytic interface may be considerably limited
by low concentrations of nitrogenous solutes in the donor
xylem stream passing through the root of the host.
The second approach, comparing mean fluxes on the
basis of surface areas of secretory pericycle parenchyma
of the nodule or of absorptive area of parenchyma at the
interface tissue of the haustorium suggests an almost
6-fold lower mean flux in nodules (0.20 /xg N mm"2 d~')
than in haustoria (1.15 /igNmm" 2 d" 1 ). However,
whereas in our calculations for the nodule, all pericycle
cells were assumed to be equally active in releasing N to
the xylem, only membranes of the outer-facing walls of
the parenchyma cells at the interface of the haustorium
were rated as participating effectively in absorbing xylem
solutes from the host root. As shown from the electron
dense tracer studies of Kuo et al. (1989), there is an
apoplastic pathway through the body of the haustorium
of Olax, and solutes carried by this avenue might well be
available for uptake by parenchyma located quite deeply
within the endophyte. Were such uptake to be appreciable,
a much greater absorptive area would be functionally
involved and estimates of mean fluxes on a membrane
area basis would have to be correspondingly reduced.
Nevertheless, the absence of lumen-lumen continuity
between host xylem vessels and xylem elements of the
haustorium (Plate ID; Kuo et al., 1989) prevents bulk
mass flow of host xylem fluid directly from host to
parasite, so the haustorium of Olax is likely to differ
radically in effectiveness of operation from that of mistletoes and certain other root hemiparasites in which lumenlumen xylem element continuities exist between partners
(Pate, 1995a, b).
The third comparison is based on mean N fluxes per
unit of lumen transectional area of xylem elements exiting
through the junction between nodule or haustorium into
the respective parent plant roots. Because of particularly
prolific xylem differentiation close to the haustorium-root
junction of Olax, the lumen transectional area for xylem
export per unit mass of haustoria turns out to be 100
times greater than that recorded for the nodule-root
junction of Acacia. This difference, taken alongside the
above-mentioned 2-fold lower throughput of N per unit
mass of haustoria than nodules, results in nodules showing
a mean exitfluxper xylem lumen area of 4891 \xg N mm"2
g F W " ' d " ' compared with only 20fxgNmm~2
g P W - i ^ - i through haustoria. This massive 200-fold
difference suggest there are inordinately greater concentrations of N in xylem conducting elements of the nodule
than in corresponding xylem of the haustorium. This is
in agreement with earlier findings demonstrating concentrations of N bleeding from xylem of detached nodules
of legumes (Pisum, Vicia) almost approaching the solubility limits of the major amino compounds which are being
transported (Pate et al., 1969; Pate and Gunning, 1972;
Gunning et al., 1974). However, in absence of direct
measurements of amounts of water passing through nodules or haustoria, N solute concentrations with the two
xylem streams cannot be assessed with any degree of
certainty. Furthermore, water flow through haustoria is
likely to be grossly affected seasonally and diurnally by
the relative transpiration rates of the shoots of the partner
species and N concentrations in the relevant xylem streams would be expected to vacillate accordingly.
Yet another form of comparison relates to the experimentally observed concentrations of N in tracheal sap
extracted from the root systems of either host or parasite
(Tables 2, 4). The values obtained (102^g Nml" 1 in
1068
Tennakoon and Pate
tracheal sap of Acacia versus 113 ^g Nml" 1 in the case
of Olax), suggest closely similar transport efficiencies in
terms of N transport per unit water flow into shoots of
the two species. These values compare fairly closely with
tracheal sap N concentrations of 109/xg Nml" 1 for
Acacia and 168 pg N ml"' for Olax obtained for similarly
pot-cultured plants used in an earlier study (Tennakoon
and Pate, 1996a), but are somewhat different from the
respective values of 86 and 204 (units as above) obtained
for Acacia and Olax, respectively, under field conditions
(JS Pate unpublished data). In the latter situation the
Olax plants sampled were parasitizing dense stands of A.
littorea and presumably benefiting almost exclusively from
this host species. In such a case higher efficiency in N
transport per unit flux of water would be anticipated.
The final assessment of mean N flux comes from
information derived from the companion study
(Tennakoon et al., 1997) on growth and partitioning of
C and fixed N in Acacia parasitized by Olax. The model
for C partitioning within the same plants as used in the
present study indicated that inputs of C through net
photosynthesis of 7136 mg by Acacia and 2957 mg by
Olax would be required to support the measured gains
of dry matter made by the partners in the study period.
Comparable cuvette based assessments of water use efficiencies were equivalent to 1.3±0.2mmol CO2 mol" 1
H2O for parasitized Acacia versus 1.0 + 0.3 mmol CO2
moP 1 H2O for Olax (Table 2 of Tennakoon et al, 1997).
So in effecting the above-mentioned dry matter gains
through net photosynthesis Acacia would be expected to
have transpired 8.23 1 of water and Olax an amount
equivalent to 4.44 1. Then, using the values for upward
xylem flow of N between root and shoot of Acacia (70 mg
N plant" 1 ) and Olax (110.5mg N plant"1) (Fig. 4 in
Tennakoon et al., 1997) one would arrive at mean concentrations in the xylem stream passing to the shoot of
Acacia of S.SmgNl" 1 versus an almost three times
greater value (24.9 mg N l -1 )for Olax. Based on such an
approach, mean xylem N concentrations in the parasite
would appear to exceed those in the host by a substantial
margin. The significance of this difference is not fully
clear.
As far as we are aware comparisons of xylem N fluxes
based on structural features of loading sites and conducting tissues have not been published elsewhere for
angiosperm parasites and their hosts. Excellent opportunities for such measurements would exist for host:mistletoe
relationships, stem and root holoparasites and other root
hemiparasites, especially where information already exists
on economies of C, N and H2O of the host(s) and
parasite (s) in question (for example, the work of Graves
et al., 1989, 1990, 1992; Cechin, 1994; Gurney et al., 1995
on Striga; that of Jeschke et al., 1994a, b on Cuscuta spp.
and a number of studies on mistletoes by Schulze et al.,
1984; Ehleringer et al., 1986; Bannister, 1989; Davidson
et al., 1989; Marshall and Ehleringer, 1990; Marshall
et al., 1994; Pate et al., 1991a, b). Structural information
would of course be needed in each case to enable fluxes
to be fully quantified for comparisons with data for other
host-parasite associations. In this manner one would
hope to provide a new dimension to the understanding
of the flow patterns and potential bottlenecks with parasitic associations and how solute flow can be engineered
so effectively in favour of the parasite through the agency
of the haustorial apparatus.
Acknowledgements
Thanks are due to Edwin Rasins and Ainsley Calladine for the
assistance provided with xylem sap analyses and image analyses
of scanned photomicrographs, respectively. This study was
supported by a grant from the Australian Research Council.
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