Journal of Experimental Botany, Vol. 47, No. 296, pp. 359-368, March 1996
Journal of
Experimental
Botany
Control of leaf expansion in sunflower
(Helianthus annuus L.) by nitrogen nutrition
S.J. Palmer1'4, D.M. Berridge2, A.J.S. McDonald3 and W.J. Davies1
1
1EBS, Division of Biological Sciences, Lancaster University, Bailrigg, Lancaster LA14YQ, UK
2
Centre for Applied Statistics, Lancaster University, Bailrigg, Lancaster LA14YF, UK
3
Department of Ecology and Environmental Research, Swedish University of Agricultural Sciences, Box 7072,
S-75007 Uppsala, Sweden
Received 24 April 1995; Accepted 1 November 1995
Abstract
Seedlings of Helianthus annuus L. were grown at an
initially high relative nitrate supply rate (0.27 mol N
mol N 1 d" 1 ). The supply was subsequently reduced
to a low rate (0.04 mol N mol N 1 d" 1 ). The response
of leaf area development to this abrupt decrease in
nitrate availability was characterized by following the
expansion of the primary and secondary leaf pairs.
The timing of the drop in nitrate supply was when cell
division in the epidermis of the primary leaf pair was
largely complete. Reducing the availability of nitrate
had a strong effect on leaf area expansion. The final
leaf size of the primary leaf pair was affected indicating an effect of nitrate availability on cell expansion.
By the end of the experiment the secondary leaf pair
was only one-third the area of that on control seedlings. The role of epidermal cell turgor pressure in this
growth response was assessed by direct measurements with a miniature cell pressure probe. No reduction in cell turgor pressure following the decrease in
nitrate availability was detected. It is concluded that
a reduction in turgor pressure was not responsible for
the reduction in leaf area expansion and it is suggested
that reduced cell expansion was due to changes in
cell wall properties. Concentrations of leaf and root
abscisic acid increased following the reduction in
nitrate availability.
Key words: Abscisic acid, cell size, cell turgor pressure,
nitrate, nitrogen, relative rate of nitrate supply.
Introduction
Studies in controlled environments and in the field have
shown that leaf area development is tightly controlled by
nitrogen availability. Plants grown at low nitrogen availability show a reduction both in leaf number and size.
The sensitivity of leaf development to nitrogen availability
is of considerable importance as productivity is often
directly proportional to leaf area development. However,
comparatively few studies have addressed the mechanism
of this response (Radin and Boyer, 1982; MacAdam
etal., 1989; Taylor et al., 1993).
The reduction in leaf size may result from reduced cell
size (McDonald, 1989), a reduction in cell division
(MacAdam et al., 1989) or a combination of the two. In
studies where a reduction in cell size has been implicated,
attempts have been made to explain this reduction in
biophysical terms (Taylor et al., 1993). The most widely
used model of cell expansion was proposed by Lockhart
(1965). Originally formulated to describe the elongation
of a cylindrical cell, the model has been widely used and
applied in studies of plant organ growth (Pritchard et al.,
1990; Serpe and Matthews, 1992). The model defines cell
expansion in terms of empirical parameters for the rate
of irreversible wall extension and the rate of water uptake.
In its simplest form the model states that cell expansion
can be described by cell turgor pressure, the extensibility
of the cell wall and the turgor pressure at which wall
yielding will occur.
Studies of nitrogen limitation to expansion have used
the Lockhart model extensively and results have been
discussed in terms of changes in parameters contained in
the model (Taylor et al., 1993; Radin and Boyer, 1982).
While doubt has recently been cast on the validity of the
model (Zhu and Boyer, 1992; Passioura and Fry, 1992),
measurement of turgor pressure remains crucial to the
interpretation of organ growth. Two schools of thought
have emerged. Radin and Boyer (1982) explained reductions in leaf area expansion by reductions in calculated
'To whom correspondence should be addressed. Fax: + 44 1524 843854. E-mail: S.Palmer©lancaster.ac.uk
C Oxford University Press 1996
360
Palmer et al.
cell turgor pressure. Other workers have shown that
calculated turgor pressure can be at least as high in leaves
of plants subjected to reduced nitrogen supply as in leaves
from plants grown at a high nitrogen supply (Taylor
et al., 1993; Stadenberg et al., 1994).
In studies of this kind the measurement of cell turgor
pressure is crucial. All previous studies with a nitrogen
variable have calculated turgor pressure from measurements of water and solute potentials in growing tissues.
Such measurements have associated difficulties and results
are open to criticism (Tomos, 1985). Here is the report
of the first study of nitrogen effects on leaf area expansion
where cell turgor pressure has been measured directly by
the use of the miniature cell pressure probe in expanding
tissues (Husken et al., 1978).
The possibility that the plant hormone abscisic acid
(ABA) is involved in the growth response (Chapin et al.,
1988) was also investigated by sampling and measurement
of leaf, root and xylem sap ABA during the experimental period.
Materials and methods
Plant culture
Seeds of Helianthus annuus cv. Tall Single Yellow (DT Brown
& Co. Ltd., Poulton-le-Fylde, Lanes., UK.) were soaked
overnight in distilled water, germinated on moist tissue paper
and sown in vermiculite. After 6 d, 32 seedlings of uniform size
(average fresh weight 0.68 g), were selected, their roots washed
free of vermiculite and transferred to a hydroponic growth unit
(Biotronic growth unit, Biotronic AB, Uppsala, Sweden).
Seedlings were supplied with a low nitrogen solution for the
first 60 h such that conductivity was between 70-80 ^S cm" 1
(pregrowth period). Uptake of nutrients tended to lower the
pH of the bathing solution which was maintained between 5.0
and 5.5 by addition of 50 mM K.OH. Temperature of the
bathing solution was maintained at 23 °C. The experiments
were conducted in a laboratory environment using continuous
light (Osram powerstar, HQ1-T, 400 W/D) with an intensity of
250 ^mol rn~ 2 s~' at plant height, air temperature 20 °C and
air humidity 30%.
The initial amount of nitrogen in the seedlings was estimated
to be 0.4% of fresh weight from measurements made on other
seedlings. After the pregrowth period during which nitrogen
stored in the seeds was depleted, nitrate was added to the
bathing solution at a rate which allowed near optimal growth
(0.27 mol N mol N ~ ' d " 1 ) . Nutrient addition was achieved by
computer-controlled operation of magnetic valves on nutrient
filled burettes. Conductivity and pH of the bathing solution
was monitored throughout the experiment. Details of nutrient
composition of the three solutions used are given in Table 1.
Nutrient addition was based on the following criteria,
1. If the pH was above the upper limit (5.5) then addition of
a complete nutrient solution of low pH was made.
2. If further nitrate was needed to meet the nitrate addition
schedule, addition of a complete neutral pH solution
was made.
3. If the conductivity of the bathing solution was below the set
limit of 70 jtS cm" 1 , then nutrients were added from a low
nitrogen solution of neutral pH.
Preliminary expenment
Plants were grown at the high rate of nitrate supply for 2
weeks. Epidermal impressions (Xantropren VL, Bayer,
Leverkussen, Germany) were taken from the adaxial surface of
the primary leaf pair on a daily basis. Six impressions were
made on each leaf, two from close to the leaf tip, two from
close to the base and two from the central portion of the leaf.
In all cases impressions were taken to the side of the main vein
in a portion of leaf blade free from larger veins. These plants
were then removed from the growth unit and leaf area was
measured using a leaf area meter (Platon Planimeter, Stepney,
Australia). Impressions were later coated with clear nail varnish.
This was allowed to dry thoroughly, peeled off the impression
and viewed through a light microscope. The number of cells in
a randomly selected field of view was counted and used to
estimate the number of adaxial epidermal cells per leaf.
Nitrogen treatments
The experiment consisted of two treatments. In the first
(control), the high rate of nutrient addition was maintained
throughout the experimental period. In the second (step
decrease), nitrate supply was dropped to a low rate (0.04 mol
N mol N " 1 d" 1 ) after the primary leaf pair had reached an
area of c. 19 cm2. This treatment was chosen in order to
separate the effects of nitrate supply on cell division from those
on cell expansion. The preliminary experiment showed cell
division in the epidermis of the primary leaf pair was complete
when leaves reached an area of approximately 16 cm2 (Fig. 1).
In this second treatment, after the supply rate of nitrate was
dropped to a low level, uptake of nutrients from the bathing
solution caused the pH to fall. As in the pregrowth period, pH
was maintained within the set limits by addition of 50 mM
KOH. At all times in the experiment, pH was maintained
between 5.0 and 5.5, while conductivity was between 70 and 80
(±S cm" 1 . The experiment was repeated on two occasions.
Measurements
Leaf length (/) and breadth (b) of the primary and secondary
leaf pair of ten plants were measured twice daily. These
measurements were used to estimate leaf area using the
relationship; area = bo + bl (Ixb); where bo = 0.31065 and b{ =
0.6413 (^ = 0.9921, n=134). Using a strip of graph paper
photocopied on acetate it was possible to measure leaf
dimensions with an accuracy of ±0.5 mm.
Adaxial epidermal cell turgor pressure was determined using
a micro-capillary pressure probe (Husken et al., 1978). For
measurement of cell turgor, plants were removed from the
growth unit. During these measurements plants were supported
in a darkened beaker filled with aerated bathing medium taken
directly from the growth unit. Direct illumination of the leaves
was avoided during turgor measurement, air temperature was
c. 24 °C and relative humidity 35%. Turgor measurements were
taken on one leaf of each plant removed from the unit, the
plant was then returned to the growth unit. Plants were never
out of the growth unit for more than 15 min in any 24 h period.
A minimum of two turgor readings was taken per leaf, with
three leaves being measured from the primary leaf pair and
three leaves being measured from the secondary leaf pair on
each sampling occasion. Output from the pressure transducer
was registered on a chart recorder. Turgor pressure measurements were only accepted if stable pressures were obtained after
insertion of the capillary or if the decline in pressure was slow
(Spollenand Sharp, 1991).
Leaf expansion
and nitrate
nutrition
361
l
Table 1. Chemical composition:
solution I and 2 equivalent to 25 g N l~ '. solution 3, 0 22 g N l~
Solution
(1) Complete (low pH)
(mmol I" 1 )
Macronutnents
K 2 SO 4
K,HPO 4
KH 2 PO 4
KNO 3
HNO3
Ca(NO 3 ) 2 .4H 2 O
Mg(NO 3 ) 2 .6H 2 O
CaCl 2 .2H 2 O
70.2
127.4
65
1418
43.7
87.5
(2) Complete (neutral pH)
(mmol I" 1 )
(3) Low N (neutral pH)
(mmol I" 1 )
70.2
70.6
56.7
121 8
64.85
108.5
36 7
387.2
431
43.7
87.5
Mga 2 .H 2 O
Micronutrients
Fe(NO 3 ) 3 .9H 2 O
Mn(NO 3 ) 2 4H 2 O
Zn(NO 3 ) 2 .4H 2 O
CuCl 2 .2H 2 O
Na 2 MoO 4 .2H 2 O
Na 2 B 4 O 7
HNO 3
3.1
1.8
1.2
0.1
0.01
3.8
2.7
3.1
1.8
1.2
0.1
001
3.8
2.7
300000
250000
V
6>
200000
8
00
0
o°
150000
0
0
100000
0
0
0
50000
10
15
20
25
30
35
40
Leaf area (cm3)
Fig. 1. Relationship between leaf area and the number of adaxial
epidermal cells. Symbols represent the mean of 8 determinations. The
arrow indicates the point at which cell division is regarded as being
largely complete.
Destructive harvests were performed on four occasions in the
experimental period. Plants were removed from the growth
unit, the stem cut above the cotyledons and xylem sap extracted
and collected by pressurizing the root stock in a Scholandertype pressure bomb. Sap was collected at 0.5 MPa, the root
stock was held in a plastic bag during collection.
Plants were then divided into root, stem primary, secondary,
and remaining leaves; these were then freeze-dried before being
weighed. Growth rates were determined on an individual plant
3.1
1.8
1.2
0.1
0.01
3.8
2.7
basis by estimation of plant weight at the previous harvest from
initial weights and average growth rate using the formula Wtx =
Wtoe"', where Wto = initial dry weight, Wt, =dry weight at time
of harvest, R = relative growth rate and / = time.
The concentration of abscisic acid in the primary and
secondary leaf pairs, roots and xylem sap was determined using
a radioimmunoassay (RIA). The monoclonal antibody (McAb)
which was specific for (+)-ABA (AFRC MAC 262), was kindly
provided by Dr SA Quarrie. ABA was extracted from leaves
and roots by shaking ground samples overnight in deionized
water in the dark at 4°C. For roots and xylem, crude extracts
were used in the RIA. For leaf extracts, sample purification
was necessary before RIA (Neales et ai, 1989). The procedure
for purification was derived from that of Subbaiah and Powell
(1987). Methanol HPLC grade (Rathburn, Walkerburn,
Scotland) and acetic acid AnalR grade (BDH, Poole, England)
were added to leaf extracts to give a ratio of 80:18:2 (by vol.)
of watenmethanokacetic acid before 1 ml samples were passed
through a C18 column (3 ml 200 mg sorbent, SepPak, Waters).
The column was then washed with water:methanol:acetic acid
(80:18:2, by vol.) and the ABA subsequently eluted with 80%
methanol 20% water buffered at pH 6-8 with ammonium
acetate. The elute was then dried in a stream of dry air,
redissolved in 0.5 ml of deionized water, acidified to pH 1.5-3.0
with hydrochloric acid and partitioned against ether. Samples
were subsequently dried, redissolved in 0.25 ml of deionized
water and subjected to analysis by RIA. Addition of internal
standards (H 3 ABA Amersham, Little Chalfont, England)
showed recovery to be c. 80%. Results were not corrected for
recovery efficiency.
Statistical analyses
The plant-specific area development of the primary leaf pair is
shown in Fig. 2. The effect of a reduction in nitrate supply
could be assessed by comparing the area of the primary leaf
pair at the end of the experiment ('final' area) in the two
treatments. However, this analysis would not utilize fully the
available data. In this paper an alternative approach has
been adopted.
362
Palmer et al.
60-
o0
i
i
2
4
6
8
10
12
14
days in growth unit
Fig. 2. Plant-specific time series of area of primary leaf pairs. Hollow
circles show the control treatment (R N = 0.27 mol N mol N " 1 d " 1 )
Filled circles: treatment where nitrate supply was stepped from a high
(R N = 0.27 mol N mol N " 1 d ' 1 ) to a low value (R N = 0.04 mol N mol
N " 1 d" 1 ) The graph uses data obtained on two occasions for
each treatment.
Figure 2 illustrates that area of the primary leaf pair followed
the same general pattern of development for all the plants.
Growth rate increased initially, then reached a maximum, and
finally tended towards zero; in other words, area eventually
would have reached a 'plateau' or asymptote which may not
have been reached by the end of the experiment. This regular
pattern of growth can be described by the logistic growth
equation (Thornley, 1990). The logistic growth equation has
been used as an efficient and effective model of growth data in
many contexts (Brody, 1968; Solomon, 1976; Ashby and
Wangermann, 1973; Dennett et al., 1978; Hunt, 1978; Thornley,
1990), and will be employed in this paper to model the
development of area of the primary leaf pair. Here the logistic
model is defined as:
afi
a-\-{p — a)e '
(1)
In general, meaningful biological interpretation may be
attached to the parameters of this model. In this context, the
parameters a, /3, and y may be interpreted as initial area,
asymptotic area, and initial specific growth rate, respectively.
Figure 2 shows that the actual pattern of growth varied from
one plant to another; the parameters in model ( I ) will differ
between plants, so model (1) was fitted to the data on a plantspecific basis. The model performed very well: each plantspecific model explained approximately 99% of the within-plant
variation.
Of the three parameters in model (1), due to the timing of
the reduction in nitrate supply only asymptotic area could have
been affected, so subsequent analyses of area development of
the primary leaf pair concentrated solely on asymptotic area.
The results of fitting model (1) were summarized in a table of
mean asymptotic areas (with corresponding standard errors),
classified by treatment and occasion (Table 2). Note that data
were collected on four occasions, with two occasions per
treatment. Asymptotic area was log transformed to reduce the
heterogeneity of variance between occasions and treatments
indicated in Table 2. Mean log asymptotic areas and corresponding standard errors are also presented in Table 2. The statistical
significance of differences in mean log asymptotic area
between treatments and between occasions (having adjusted for
treatment) was tested with an analysis of variance (ANOVA
1, Table 3).
This analysis of area development of the primary leaf pair
maximized data utility, while simultaneously reducing the
dimensionality of area growth data to a single biologically
meaningful response, namely asymptotic area.
The adaxial epidermal cell turgor pressures observed on the
two occasions when the nitrate supply was reduced were
summarized in a two-way table of means cross-classified by
level of nitrate supply (high/low) and location (primary/
secondary leaf pair) (Table 4). ANOVAs taking the different
levels of variation within the pressure data into account were
performed for each treatment separately. Time was handled as
a continuous covariate in the ANOVA on the control treatment
(ANOVA 2, Table 5) in order to investigate whether there was
evidence of an underlying linear trend in mean cell turgor
pressure over time. Level of nitrate supply was treated as a
factor at two discrete levels in the ANOVA on the step decrease
treatment (ANOVA 3, Table 6) in order to assess the statistical
significance of a change in mean cell turgor pressure after the
reduction in nitrate supply. If there proved to be no statistically
significant underlying trend over time in ANOVA 2, then any
within-plant differences that were statistically significant in
ANOVA 3 could be attributed to the reduction in nitrate supply.
Results
Preliminary experiment
The relationship between leaf area and number of adaxial
epidermal cells shows two clear phases. In the first phase
until leaves reach c. 16 cm2 cell number increases with
leaf area. After this leaf area can be seen to increase
without any further increase in cell numbers. In this way
the expansion of the primary leaf of sunflower can be
separated into a period of cell expansion and cell division
followed by a period dominated by cell expansion.
Nitrogen treatments
In the control treatment after the pregrowth period,
relative growth rate approximated the addition rate of
nitrate (Fig. 3). In the step decrease treatment relative
growth rate decreased after the supply of nitrate was
reduced.
Figure 4 shows the area development of the first two
leaf pairs over the experimental period, aggregated across
plants. On three of the four occasions, the aggregate area
of all primary leaf pairs had reached its asymptote by the
end of the period (Fig. 4A, B). The secondary leaf pairs
were still expanding at the end of all four occasions
(Fig.4C, D).
Figure 4A and B clearly show that reducing the nitrate
supply had an effect on area expansion in the primary
Leaf expansion and nitrate nutrition
363
Table 2. Mean asymptotic area, and log asymptotic area, of primary leaf pairs ± SE
Treatment
Occasion
Means
Treatment
Means
Control
Step decrease
Occasion
Occasion
Al
Bl
A2
B2
38.84 + 3.63
3.622±O.O91
38.13±2.35
3.602±0.066
37.43±3.16
3 582±O.1OO
33.49 ±1.89
3.498 ±0 054
28.68 ±1.50
3.332 ±0.050
23.87±0.91
3.166±0.038
Table 3. Analysis of variance of log asymptotic area of primary
leaf pairs (AN OVA 1)
Source of
variation
Degree of
freedom
Treatment
1
Occasion, after
adjusting
for treatment 2
Residual
36
39
Total
Sum of
squares
Mean
square
Ratio of mean
squares (m.s.r.)
0 73065
0.73065
12.82**
0.55779
2.05130
3.33974
0.27889
0.05698
4.89*
•Significant at 5% level (/><0.05).
** Significant at 1% level (/><0.01).
leaf pair. This is further demonstrated by a comparison
of the mean asymptotic area of the primary leaf pair in
the two treatments (Table 2). On the evidence of the
growth patterns observed during the experiment, primary
leaves from plants in the control treatment would have
had approximately four-thirds the asymptotic area of
those from plants experiencing the reduction in nitrate
supply. This difference was statistically significant at the
1% level (Table 3).
Expansion of the secondary leaf pair, which was less
developed at the time of reduction in nitrate supply, was
affected to a greater extent. By the end of the experiment
leaves from the control treatment were approximately
three times the area of those from plants which experienced the reduction in nitrate supply. It is clear from
Fig. 4C and D that this difference was highly significant.
Figure 5 shows the adaxial epidermal cell turgor pressures for the first two leaf pairs. There was no evidence
of a statistically significant linear trend in mean cell turgor
pressures over time during the control occasions (Table 5:
time m.s.r. <1, P>0,05). However, following the reduc-
tion in nitrate supply, there was a small increase in mean
cell turgor pressure (Table 4) which was statistically significant at the 1% level (Table 6: level m.s.r. = 20.04,
P<0.0\). The difference in mean cell turgor pressure
between the primary and secondary leaf pairs was statistically significant in both treatments (Table 5: location
m.s.r. =4.99, P<0.05; Tables 4 and 6: location m.s.r. =
15.03, P<0.0\). This difference changed significantly
from one control occasion to another (Table 5: occasion
by location m.s.r. = 5.52, P<0.05), and also when the
nitrate supply was reduced (Tables 4 and 6: location by
level m.s.r. =.10.91, P<0.0\).
Figures 6 and 7 show root and leaf ABA concentration.
In the control treatment both root and leaf ABA concentrations remained relatively stable through the experimental period. Dropping the nitrate supply rate resulted
in an increase in both leaf and root ABA concentration.
At the end of the experimental period both leaf and root
ABA concentrations were c. 3 times higher than in the
control treatment. Xylem ABA concentration remained
stable through the experimental period (data not shown)
and was unaffected by the reduction in nitrate supply
(Table 7).
Discussion
Where nutrients are taken up in adequate proportions
and in exponentially-increasing amounts plant relative
growth rate has been found to equal the relative rate of
increase in the most limiting nutrient (Ingestad, 1982;
Ingestad and Agren, 1992). In the present experiment,
the addition procedure was such that nitrate was the most
limiting nutrient to plant growth.
Table 4. Mean adaxial epidermal cell turgor pressure ± SE: two step decrease occasions
Location means
Level of nitrate supply
Location
Primary
Secondary
Level of nitrate supply means
High
Low
0.4746 ±0.0053
0.4896 ±0.0090
0.4772 ±0.0047
0.4902 ±0.0059
0.5463 ±0.0070
0.5169 ±0.0053
0.4808 ±0.0040
0.5313 ±0.0064
364
Palmer et al.
Table 5. Analysis of variance of adaxial epidermal cell lurgor pressure: two control occasions (ANOVA 2)
Source of
variation
Sum of
squares
Mean
square
Ratio of mean
squares (m.s.r.)
1
35
36
0.001695
0.150416
0.152111
0.001695
0.004298
<1
1
1
12
14
0.010410
0.011501
0.025020
0.046931
0.010410
0.011501
0 002085
1
1
1
1
116
170
0.000099
0.001196
0.0O0359
0.000102
0.174151
0.374949
0000099
0.001196
0.000359
0.000102
0.001501
Degree of
freedom
Between plant variation
Occasion
Residual
Sub-total
Between leaf variation
Location
Occasion by location
Residual
Sub-total
Within plant variation
Time
Occasion by time
Location by time
Occasion by location by time
Residual
Grand total
4.99*
5.52*
<1
<1
<!
<1
•Significant at 5% level (/*<0.05).
Table 6. Analysis of variance of adaxial epidermal cell turgor pressure: two step decrease occasions (A NOVA 3)
Source of
variation
Between plant variation
Occasion
Residual
Sub-total
Between leaf variation
Location
Occasion by location
Residual
Sub-total
Within plant variation
Level of nitrate supply
Occasion by level
Location by level
Residual
Grand total
Degree of
freedom
Sum of
squares
Mean
square
Ratio of mean
squares (m.s.r.)
1
39
40
0.000930
0.162790
0.163720
0.000930
0.004174
<1
1
1
13
15
0.073340
0.000238
0.063441
0.137019
0.073340
O.OOO238
0.OO4880
15.03**
<1
1
1
1
148
206
0.035226
0.000140
0.019185
0.260151
0.615441
0.035226
0.000140
0.019185
0.001758
20.04**
<1
10.91**
'Significant at 1% level (/><0.01).
The expected equality of growth and supply rates was
obtained when the supply rate was maintained high
(Fig. 3). However, following the step decrease in nitrate
supply, the equality of growth rates and supply rates was
not apparent. It is assumed that the plants were in a lag
phase of acclimation to the new supply rate of nitrogen
(Ingestad and Lund, 1979). Through the experimental
period, growth rates tended towards the new supply rate.
The effect on the expansion of the primary leaf pair
suggests that in Helianthus nitrate supply affects cell
expansion. This finding is supported by studies on Salix
viminalis (McDonald, 1989; Stadenberg et al., 1994)
which showed reduced epidermal cell size at low rates of
nitrate supply. However, MacAdam et al. (1989) found
nitrogen supply affected the rate of production of cells
and not their expansion in leaf blades of tall fescue
(Festuca arundinacea Schreb.). This difference in response
may be attributable to species and it may be informative
to compare the response of a number of monocotyledons
and dicotyledons.
While this experiment was not designed to test the
speed of response of leaf area increase to a reduction in
nitrate supply, the results suggest that this is a quick
response. Even with the relatively crude measurement
technique employed, the reduction in leaf area expansion
is apparent within 24 h.
Little variation in cell turgor pressure was found in
this study, either between cells, leaves or plants. Variation
might be expected to be less in this study than with soilgrown plants as the supply of water to the roots is
constant with no complications of localized soil drying.
Although cell turgor pressure was only measured in the
epidermis, in studies of stems and coleoptiles where it
was possible to determine the expansive properties of
Leaf expansion and nitrate nutrition
365
A.
0.40
0.35 -
B.
0.00
5
10
days in growth unit
Fig. 3. Time series of relative rates of increase in plant DW. Nitrate
treatments: RN = 0.27 mol N mol NT 1 d" 1 (O); RN = 0.27 mol N mol
r - r l d ~ ' , then RN = 0.04 mol N m o l N " 1 d" 1 after arrow ( • ) . Symbols
represent the mean of 8 determinations. Bars indicate SE. The dashed
lines indicate the rate of nitrate supply.
0
3
6
9
12
days in growth unit
0
3
6
S
12
15
18
0
3
days in growth untt
80
B
12
15
18
day* In grow* unit
c.
100
6
Fig. 5. Time series of adaxial cell turgor pressure. (A) RN = 0.27 mol N
mol NT 1 d" 1 ; (B) RN = 0.27 mol N mol 1ST1 d" 1 , then RN = 0.04 mol
N mol NT 1 d" 1 after arrow; primary leaf pair (O), secondary leaf pair
(V). The graph uses data obtained on two occassions for each treatment.
D.
:
100 -
i
I-
$
* 4
V
V
20
0
3
8
9
12
days In growfli unit
15
18
0
3
6
9
12
15
18
days In growth unrt
Fig. 4. Time series of leaf area. (A, B) Primary leaf pair, (C, D)
secondary leaf pair. RN = 0.27 mol N mol N " 1 d" 1 (O V); RN = 0.27
mol N mol N " ' d ~ \ then RN = 0.04 mol N mol N ' 1 d" 1 after arrow
( • • ) . Symbols represent mean values of 10 measurements. Bars
indicate SE.
different tissues, the epidermis has been found to limit
organ expansion (Fry, 1988; Hodick and Kutschera, 1992;
Kutschera, 1992).
No reduction in cell turgor pressure was observed
following the step-decrease in nitrate supply. Previous
studies have shown that where plants are grown in soil
6
9
12
15
18
days In growth unit
Fig. 6. Time series of root ABA concentration. RN == 0.27 mol N mol
NT 1 d" 1 (O) and RN = 0 27 mol N mol N " 1 d"'
N mol N " 1 d" 1 after arrow ( • ) . Symbols represent the mean of 6 to
9 determinations. Bars indicate SE.
366
Palmer et al
6
6
9
12
15
18
days In growth unit
Fig. 7. Time series of leaf ABA concentration. Hollow symbols RN =
0.27 mol N mol N " 1 d" 1 ; symbols filled: RN = 0.27 mol N mol N " 1
d " ' , then RN = 0.04 mol N mol N " ' d ~' after arrow; primary leaf pair
( O , # ) , secondary leaf pair (V, Y). Symbols represent the mean of at
least 6 to 9 determinations. Bars indicate SE
Table 7. Concentration of xvlem ABA ±SE
Treatment
ABA concentration
(f*m m )
Sample number
Control
Step decrease
2.161 ±0.119
2 158±0.193
19
17
or 'traditional' nutrient systems nitrate is an important
osmoticum. For example, the study of Fricke et al. (1994)
showed that nitrate comprised some 36% of the total
solutes in the epidermis of the third leaf of barley. That
cell turgor pressure does not fall in response to the abrupt
drop in nitrate supply rate indicates either that nitrate
has been replaced as a major osmoticum by another
anion, or that nitrate is not a major osmoticum when
plants are grown using the system used in this experiment.
Flexibility of epidermal anion composition has been suggested by Fricke et al. (1994) and is supported by the
work of Pope and Leigh (1990) who concluded that in
red-beet NO 3 " and Cl" were transported competitively by
the same transporter into the vacuole.
In contrast to results reported here, Radin and Boyer
(1982) were able to detect reduced turgor pressure in
sunflower plants deprived of nitrogen. However, their
measurements of turgor pressure were obtained by calculating turgor pressure from values of water and solute
potentials measured with isopeistic psychrometry on
excised tissue. This calculation of turgor pressure has
recognized problems. Firstly, on excision of growing
tissue, cells will continue to expand and turgor will fall
(Cosgrove, 1993). Thus, calculated turgor will be lower
than in vivo turgor. Furthermore, water potential measurements are made at equilibrium so the importance of
hydraulic conductance in the growing tissue may be
missed. There is also the problem of mixing of cell
contents with apoplastic water and solutes. These problems mean that in order to determine the role of turgor
pressure in a growth response, direct measurements of
the kind obtained in this experiment are to be preferred.
Radin and Boyer (1982) followed up their observations
of reduced turgor pressure in nitrogen-deprived plants by
measuring the flow rate of sap through pressurized root
systems. They were able to show treatment differences
which they argued reflect differences in root hydraulic
conductivity. In this way they concluded that nitrogen
deprivation was affecting cell turgor pressure (and hence
leaf growth) through an effect on root hydraulic conductivity. However, if nitrogen deprivation was affecting root
hydraulic conductivity on a time-scale necessary to
explain the reduction in leaf area expansion reported in
this study, then a reduction of cell turgor pressure should
have been observed, which was not the case. In this study
plants from both treatments had high rates of conductance (c. 200 mmol m - 2 „ - ! measured with diffusion
porometer Mark 4, Delta T Devices Ltd), so an increase
in the pathway resistance to water flow through the plant
would be expected to reduce cell turgor pressure. It is
concluded that, in the time span of this study, reducing
nitrate availability had no significant effect on root
hydraulic conductivity.
Because cell turgor pressure did not fall, it is proposed
that the drop in nitrate supply reduced cell expansion by
affecting cell wall properties. This is supported by the
small, but statistically significant, increase in mean turgor
pressure recorded following the drop in nitrate supply.
The finding that cell wall properties are limiting expansion
rate is in agreement with a number of other studies (Zhu
and Boyer, 1992; Serpe and Matthews, 1992; Pritchard
et al., 1990). Although the control of cell wall expansion
remains poorly understood, recent work has focused on
the role of enzymes which cleave matrix polymers (Inouhe
and Nevins, 1991; Smith and Fry, 1991) and proteins
which facilitate cell expansion (McQueen-Mason et al.,
1992). In order for such enzymes to provide a dynamic
control of cell expansion, their turnover or activity must
be tightly regulated. This may be the point at which
nitrogen affects cell expansion. This effect may be specific
by affecting the turnover or activity of enzymes or proteins
controlling cell expansion, analogous to the specific effects
of nitrate in the regulation of nitrate reductase (Shaner
and Boyer, 1976, and see review by Solomonson and
Barber, 1990). The possibility therefore exists that nitrate
induces the activity of key enzymes involved in cell wall
relaxation. However, the effect of nitrate on cell expansion
may be of a less specific nature affecting enzyme activity
Leaf expansion and nitrate nutrition
in a more general way (Paul and Stitt, 1993). In this
instance the tight linking of nitrate availability and leaf
expansion would be a consequence of the fast turnover
of key enzymes or proteins involved in cell wall expansion
or the control of such enzymes.
The results of our study confirm previous results which
have shown an increase in abscisic acid concentration at
low nitrate availability (Chapin et al., 1988). Transient
effects on root zeatin riboside concentration following
changes in the supply of nitrate have also been reported
(Samuelson et al., 1992) and an effect of nitrate availability on the export of cytokinin from the root has been
shown (Wagner and Beck, 1993). The increase in ABA
concentration together with the reports of transient
decreases in cytokinin concentration indicate that the
hormonal balance of plants is shifted if nitrate is withheld.
Whether these changes are consequences or causes of the
changes in plant growth is not known. The reduction in
leaf expansion rate when nitrate supply is dropped is
further complicated by the possibility of an interaction
of nitrate supply with plant growth regulators, in that
alterations in plant growth regulator concentration may
provide a secondary signal of nitrate availability.
In conclusion, this experiment has clearly demonstrated
a tight temporal coupling of leaf expansion and nitrate
supply which was not mediated via leaf turgor pressure.
Acknowledgements
The authors would like to thank Finn Povlsen for his help with
purification of ABA samples and Stuart Thompson for his help
and advice with pressure probing.
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