Directory UMM :Data Elmu:jurnal:T:Tree Physiology:vol17.1997:
Tree Physiology 17, 97--103
© 1997 Heron Publishing----Victoria, Canada
Gas exchange and water relations ofFraxinus americana affected by
flurprimidol
GNANASIRI S. PREMACHANDRA, WILLIAM R. CHANEY and HARVEY A. HOLT
Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN 47907, USA
Received August 30, 1995
Summary Effects of flurprimidol on plant water relations
and leaf gas exchange were investigated in one-year-old white
ash (Fraxinus americana L.) seedlings subjected to soil water
deficits. Flurprimidol (20 mg kg −1 of soil equivalent) was
applied to the soil surface of pot-grown seedlings after shoot
growth was completed. Two months after flurprimidol application, water was withheld from one-half of the seedlings. Leaf
water relations and gas exchange parameters were measured 5,
7, 10, 14, 18 and 22 days after withholding water. Under both
irrigated and nonirrigated conditions, flurprimidol treatment
resulted in reduced net CO2 assimilation rate and transpirational water loss of seedlings as a result of decreased stomatal
conductance. Consequently, flurprimidol-treated seedlings had
higher leaf water potential and relative water content than
untreated seedlings. Nonirrigated flurprimidol-treated seedlings also had greater turgor and sap osmolality and lower
osmotic potential at full turgor than seedlings in the other
treatments, indicating that flurprimidol increased osmotic adjustment. Under water-stress conditions, water use efficiency
was lower and gas exchange efficiency was higher in flurprimidol-treated seedlings than in untreated seedlings, suggesting
that flurprimidol treatment enhances survival of plants subjected to soil water deficits.
ery stock with a growth inhibitor may improve plant survival
and quality at times when water is limited.
Flurprimidol (α-(1-methylethyl)-α-[4-(trifluoromethoxy)
phenyl]-5-pyrimidine-methanol), a gibberellin synthesis inhibitor (Rademacher 1991), is used to control shoot growth in
trees. Flurprimidol and other pyrimidine PGRs modify the
growth of plants by causing shorter internodes and smaller
leaves (Davis and Curry 1991). In addition to interfering with
gibberellin biosynthesis, pyrimidine compounds also promote
the biosynthesis of abscisic acid (ABA) (Cowan and Railton
1987, Grossmann 1992). Both abscisic acid and gibberellins
are synthesized via the isoprenoid pathway, and the two plant
hormones often exhibit opposing physiological activities
(Loveys and Milborrow 1984). The role of ABA in stomatal
functioning and plant water relations is well documented (Kozlowski et al. 1991). Although the growth regulating effects of
flurprimidol have been investigated in several herbaceous species, little is known about its effects on the physiological
processes of tree species. Therefore, we have studied the effects of flurprimidol on the plant water relations, leaf gas
exchange, and water use efficiency of irrigated and nonirrigated white ash (Fraxinus americana L.) seedlings to test the
hypothesis that flurprimidol treatment enhances plant tolerance to water stress.
Keywords: osmotic adjustment, plant growth regulators, water
stress.
Materials and methods
Introduction
Several plant growth regulators (PGRs) are used to suppress
shoot growth in horticultural crops and urban trees (Davis and
Curry 1991). In addition to controlling growth, PGRs also
increase root/shoot ratios (Early and Martin 1988, Numbere et
al. 1992), decrease stomatal conductance (Armitage et al.
1984, DeJong and Doyle 1984, Steinberg et al. 1991a), increase root hydraulic conductivity and xylem pressure potential (Vaigro-Wolff and Warmund 1987, Rieger and Scalabrelli
1990) and decrease plant water use by reducing transpiration
(Atkinson and Chauhan 1987, Schuch 1994). Because all of
these effects tend to make plants more drought tolerant and
improve their water use efficiency, Frymire and HendersonCole (1992) suggested that treatment of container-grown nurs-
One-year-old seedlings of white ash were obtained from the
Indiana State Tree Nursery and graded to uniform size. On
April 10, 1994, each of 100 seedlings was planted in a 15.2-liter plastic pot containing a 7/3/2 (v/v/v) mix of top soil/peat
moss/perlite amended with (per m3): 680 g Ca(H2PO4)2, 454 g
KNO3, 454 g MgSO4, 3.6 kg ground limestone and 57 g fritted
trace elements. The seedlings were grown in a greenhouse at
Purdue University, West Lafayette, Indiana, USA, in a natural
photoperiod at a maximum day/minimum night temperature of
approximately 28/23 °C until terminal shoot growth was completed and the leaves were fully expanded.
After leaf growth ceased in early June, one half of the
seedlings were randomly selected for flurprimidol treatment.
On June 14, 1994, flurprimidol was applied to the soil at the
rate of 20 mg kg −1 of soil (312 mg flurprimidol per15.6 kg soil
in each pot). No additional leaf flushes occurred in either
98
PREMACHANDRA, CHANEY AND HOLT
flurprimidol-treated (flur(+)) or untreated (flur(−)) seedlings.
On August 11, 1994, two months after flurprimidol application, one half of the flurprimidol-treated and untreated seedlings were randomly selected for water stress treatment. They
were not watered again for the 22-day duration of the study.
The other half of the treated and untreated seedlings continued
to receive regular watering to resaturation every other day and
served as irrigated controls.
Leaf water relations and gas exchange parameters were
measured to compare flurprimidol-treated and untreated seedlings under irrigated and nonirrigated conditions 5, 7, 10, 14,
18 and 22 days after water was withheld. Before dawn on the
day of measurement, 20 seedlings (five replicates from each of
the four treatments) were moved to a large growth chamber
where sampling could be carried out under conditions of uniform light, temperature and humidity. Light from six 1000-W
high-pressure sodium vapor lamps (Energy Technics, York,
PA) was filtered through a 3-cm layer of water, providing an
incident photosynthetic photon flux to the sample leaves of
approximately 900 µmol m −2 s −1. Temperature and relative
humidity were 25 ± 2 °C and 40 ± 5%, respectively, and
ambient CO2 concentration was 340--360 µl l −1. Plants were
equilibrated under these conditions for 3 h before measurements were initiated.
Measurement of leaf gas exchange
Net CO2 assimilation rate (A), stomatal conductance (gs), transpiration rate (E) and internal CO2 concentration (Ci) were
measured between 1000 and 1200 h on two leaflets of the two
uppermost, fully expanded leaves with an ADC portable photosynthetic and transpiration measurement system (Analytical
Development Company Limited, Hoddesdon, England). Instantaneous water use efficiency (WUE) was calculated as the
ratio of A/E (mmol mol −1). Intrinsic gas exchange efficiency
(GEE) was calculated as the ratio of A/gs (µmol mol −1).
Measurements of leaf water potential, osmotic potential and
relative water content
Discs (0.5-cm diameter) were cut from the same leaflets used
for gas exchange measurement and immediately put in thermocouple psychrometer sample chambers (Wescor C-52; Wescor
Inc., Logan, UT) and allowed to equilibrate for 4 h. Leaf water
potential (Ψw) was then measured psychrometrically with a
microvoltmeter. The Ψw values were accurate to ± 0.05 MPa
and were comparable to Ψw values measured with a pressure
chamber. Afterwards, the leaf discs were frozen and thawed in
the psychrometer sample chambers and their osmotic potential
(Ψπ) was measured psychrometrically. Leaf Ψπ was corrected
for the dilution of symplastic sap by apoplastic water, which
occurs when cell walls are broken during freezing and thawing
of leaf discs (Tyree 1976); a constant apoplastic fraction of
0.16 was assumed (Parker and Pallardy 1987). Turgor (Ψp) was
calculated by subtracting the corrected Ψπ from Ψw. Relative
water content (RWC) was computed as RWC = (FW--DW)/
(TW--DW) (where FW = fresh weight, DW = dry weight and
TW = turgid weight), using 1-cm-diameter discs excised from
leaflets of the same two leaves used for previous measure-
ments. Osmotic potential at full turgor (Ψπ(100) ) was calculated
as Ψπ(100) = Ψπ (RWC − AWC)/(1.0 − AWC), where AWC is the
apoplastic water content (Wilson et al. 1979). Osmotic adjustment (OA) was estimated as the difference between the Ψπ(100)
values estimated in leaves of irrigated and nonirrigated plants.
Measurement of sap osmolality
Several leaflets from the two leaves that were used for gas
exchange and water relations measurements were frozen in
sealed polyethylene freezer bags. Leaf samples were thawed
and centrifuged at 1000 g for 20 min at 6--8 °C to extract cell
sap. Sap osmolality was measured with a Wescor Model
5100C vapor pressure osmometer (Wescor Inc.) and was corrected for dilution of symplastic sap by apoplastic water.
Statistical analyis
Data were subjected to analysis of variance. For the first four
measurement periods, a 4 × 2 × 2 × 5 factorial design was used,
including four sampling periods, two flurprimidol treatments,
and two watering treatments with five replications. Because
many of the nonirrigated flur(−) seedlings died between sampling days 14 and 18, no measurements were made of seedlings in this treatment for sampling days 18 and 22. Data for
flur(+), nonirrigated seedlings were analyzed in a 6 × 5 completely randomized block design with six sampling periods
and five replications. The least significant difference (LSD) at
the 0.05 level was calculated to determine differences among
means. Regression analyses of the various parameters measured as a function of leaf Ψw were performed and best fit curves
plotted.
Results
Figures 1 and 2 illustrate changes in leaf Ψw, RWC, Ψp, Ψπ(100),
and OA of flurprimidol-treated (flur(+)) and untreated (flur(−))
white ash seedlings under irrigated and nonirrigated conditions
over time. For the first 14 days after withholding water, leaf Ψw
was higher in flur(+) seedlings than in flur(−) seedlings under
both irrigated and nonirrigated conditions (Figure 1A). Leaf
Ψw of irrigated seedlings was 0.21 to 0.36 MPa higher in the
flur(+) treatment than in the flur(−) treatment during this
period. In the nonirrigated treatments, Ψw decreased more
rapidly in flur(−) seedlings than in flur(+) seedlings (−5.09
versus −2.13 MPa on Day 14) (Figure 1A). Leaf Ψw of nonirrigated flur(+) seedlings reached −3.43 and −5.00 MPa 18 and
22 days after withholding water, respectively. Leaves for
which a Ψw value of −5 MPa was measured were not severely
wilted, presumably because of their high cellulose content, but
the leaf margins and small areas of the leaves were beginning
to turn brown.
Relative water content in nonirrigated flur(−) seedlings decreased rapidly after water was withheld, declining from 81.5
to 53.6% between Days 5 and 14. The corresponding RWC
values for nonirrigated flur(+) seedlings were 87.7 to 83.7%.
With further exposure to water stress, RWC in nonirrigated
flur(+) seedlings decreased to 63.5 and 43.1% by Days 18 and
22, respectively (Figure 1B).
FLURPRIMIDOL IMPROVES TOLERANCE TO WATER STRESS
Under both irrigated and nonirrigated conditions, turgor was
significantly greater in flur(+) seedlings than in flur(−) seedlings, except in irrigated seedlings on Days 7 and 14 (Figure
2A). Irrigated flur(+) seedlings exhibited a Ψp of 2.01 to 2.25
MPa compared with 1.69 to 2.13 MPa for irrigated flur(−)
seedlings. Turgor of nonirrigated flur(−) seedlings decreased
with time and reached zero at Day 14. Nonirrigated flur(+)
seedlings maintained higher Ψp at lower leaf Ψw and for a
longer time than flur(−) seedlings, exhibiting a Ψp of 0.4 MPa
even after water was withheld for 22 days (Figures 2A and 3A).
In both flur(−) and flur(+) seedlings, withholding water for
14 days resulted in a significant decrease in Ψπ(100) (Figure 2B). Osmotic potential at full turgor of nonirrigated flur(+)
seedlings decreased by 0.6 MPa from Days 5 to 18. At Day 22,
Ψπ(100) of nonirrigated flur(+) seedlings was 0.2 MPa above
that at Day 5 (Figure 2B). Nonirrigated flur(+) seedlings maintained a lower Ψπ(100) at a given leaf Ψw than flur(−) seedlings,
except at Ψw values below −5 MPa (Figure 3B).
In the nonirrigated treatments, osmotic adjustment was evident in both flur(−) and flur(+) seedlings (Figure 2C). The
greater osmotic adjustment in flur(−) seedlings than in flur(+)
seedlings at 10 and 14 days after withholding water was a
result of exposure of flur(−) seedlings to a higher degree of
water stress as measured by Ψw. Sap osmolality was greater in
flur(+) seedlings than in flur(−) seedlings over a wide range of
leaf Ψw values (Figure 3C).
The effects of irrigation treatment on A and gs were observed
as soon as 5 days after withholding water in flur(−) seedlings;
however, in flur(+) seedlings, the effects were observed only
during the later stages of stress development (Figures 4A and
4B). For example, in flur(+) seedlings, there were no differ-
Figure 1. Leaf water potential (A) and relative water content (B) of
flurprimidol-treated (Flur+) and untreated (Flur−) white ash seedlings
grown in irrigated (Irri) or nonirrigated (Non) conditions at 5, 7, 10,
14, 18 and 22 days after withholding water. The LSD is at 0.05 level.
99
ences in A and gs between nonirrigated and irrigated seedlings
until Days 14 and 10, respectively. Net CO2 assimilation in
irrigated flur(+) seedlings was about 50% of that in irrigated
flur(−) seedlings. Similarly, gs was lower in irrigated flur(+)
seedlings than in irrigated flur(−) seedlings. In flur(−) seedlings, both A and gs decreased rapidly with increasing water
stress and reached values that were 87 and 89% lower on Days
5 and 14, respectively, than in irrigated flur(−) seedlings. Net
CO2 assimilation rate in nonirrigated flur(+) seedlings declined by 93%, but only after withholding water for 22 days.
Likewise, stomatal conductance in nonirrigated flur(+) seedlings declined by 95% between Days 5 and 22. Flur(+) seedlings exhibited lower A and gs than flur(−) seedlings over a
wide range of leaf Ψw values (Figures 5A and 5B).
Transpiration rate was lower in nonirrigated than in irrigated
seedlings in both flur(+) and flur(−) treatments at all sampling
periods up to 14 days after withholding water (Figure 4C).
Flurprimidol reduced E by 45% in irrigated seedlings. In
nonirrigated seedlings, flur(+) seedlings exhibited a lower E
than flur(−) seedlings up to 7 days after withholding water, and
thereafter, flur(+) seedlings exhibited a higher E than flur(−)
seedlings. Transpiration rates of nonirrigated flur(+) seedlings
on Days 18 and 22 were approximately 53 and 12%, respectively, of those on Day 5. Nonirrigated flur(+) seedlings had a
lower E than flur(−) seedlings over a wide range of leaf Ψw
Figure 2. Turgor (A), osmotic potential at full turgor (B) and osmotic
adjustment (C) of flurprimidol treated (Flur+) and untreated (Flur−)
white ash seedlings grown in irrigated (Irri) or nonirrigated (Non)
conditions at 5, 7, 10, 14, 18 and 22 days after withholding water. The
LSD is at 0.05 level.
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PREMACHANDRA, CHANEY AND HOLT
Figure 3. Regression of turgor (A), osmotic potential at full turgor (B)
and sap osmolality (C) as functions of leaf water potential in flurprimidol-treated (Flur+) and untreated (Flur−) white ash seedlings. Variables were measured in leaves of container-grown seedlings during a
22-day period in which water was withheld. The Flur+ and Flur−
treatment effects were significantly different (P = 0.05).
values (Figure 5C). Neither the flurprimidol treatments nor the
irrgation treatments affected Ci (Figure 4D).
The effects of a range of leaf Ψw values on water use
efficiency and gas exchange efficiency of flur(+) and flur(−)
white ash seedlings are shown in Figure 6. Water use efficiency
of flur(+) seedlings was lower than that of flur(−) seedlings at
leaf Ψw values ≤ −2 MPa, except at Ψw = −5 MPa, where they
were similar. Flur(+) seedlings had a higher WUE than flur(−)
seedlings at high leaf Ψw (−1 MPa). In contrast, GEE was
significantly greater in flur(+) seedlings than in flur(−) seedlings only when leaf Ψw was less than −2 MPa.
Discussion
Flurprimidol decreased stomatal conductance in white ash
seedlings even when soil water was not limiting. On all measurement days, stomatal conductance in irrigated flur(+) seedlings was lower than in irrigated flur(−) seedlings, resulting in
lower A and E. In the nonirrigated treatments, gs of flur(+)
seedlings was only reduced below that of flur(−) seedlings
Figure 4. Net CO2 assimilation rate (A), stomatal conductance (B),
transpiration rate (C) and internal CO 2 concentration (D) of flurprimidol-treated (Flur+) and untreated (Flur−) white ash seedlings grown in
irrigated (Irri) or nonirrigated (Non) conditions for 5, 7, 10, 14, 18 and
22 days after withholding water. The LSD is at 0.05 level.
14 days after withholding water. Nonirrigated flur(+) seedlings had higher Ψw, Ψp, and leaf RWC than nonirrigated
flur(−) seedlings, and only the flur(+) seedlings survived for
more than 14 days after water was withheld, presumably as a
result of reduced E, reduced plant water use, and increased
turgor maintenance. Osmotic adjustment occurred in flur(−)
seedlings on Days 10 and 14, and occurred in flur(+) seedlings
on Day 14, as indicated by decreased Ψπ(100) and increased sap
osmolality.
The values of Ψw (−5 MPa) determined for leaves of nonirrigated seedlings are low compared with published values. The
leaves of most vascular plants are irreversibly damaged at such
low water potentials as a result of mechanical tearing of protoplasm and degradation of membranes under the extreme tensions developed within cells (Gaff 1980). For example, Cleary
and Zaerr (1980) reported that vigor of ponderosa pine seedlings declined progressively between Ψw values of −2 and
−5 MPa, and the seedlings died when Ψw reached −5 MPa.
FLURPRIMIDOL IMPROVES TOLERANCE TO WATER STRESS
101
Figure 6. Regression of water-use efficiency (A) and intrinsic gas-exchange efficiency (B) as functions of leaf water potential in flurprimidol-treated (Flur+) and untreated (Flur−) white ash seedlings during a
22-day period in which water was withheld. Water-use efficiency was
calculated as the ratio of net CO2 assimilation rate/transpiration rate,
and intrinsic gas-exchange efficiency was calculated as the ratio of net
CO2 assimilation rate/stomatal conductance. The Flur+ and Flur−
treatment effects were significantly different (P = 0.05).
Figure 5. Regression of net CO2 assimilation rate (A), stomatal conductance (B) and transpiration rate (C) as functions of leaf water
potential in flurprimidol-treated (Flur+) and untreated (Flur−) white
ash seedlings. Variables were measured in leaves of container-grown
seedlings during a 22-day period in which water was withheld. The
Flur+ and Flur− treatment effects were significantly different
(P = 0.05).
There are few studies on the effects of flurprimidol or other
pyrimidine growth inhibitors on plant water relations and gas
exchange. Pyrimidine growth inhibitors reduce water use
(Johnson 1974, Barrett and Nell 1981), and the reduction is
partially related to the smaller leaf area of the treated plants
(Davis et al. 1986, Vaigro-Wolff and Warmund 1987, Sterrett
et al. 1989). Barrett (1983) reported a reduction of 35 and 39%
in total plant transpiration in poinsettias treated with EL-500
(flurprimidol) and ancymidol, respectively, whereas on a per
leaf basis the difference in transpiration was only 12% for both
compounds.
High root/shoot ratios are normally observed in PGRtreated plants (Early and Martin 1988, Numbere et al. 1992),
and could lead to improved water absorption and water availability to shoots (Steinberg et al. 1991b). However, in the
present study, the flurprimidol treatment was applied after
terminal shoot growth was completed and leaves were fully
matured. Hence, there was no new shoot growth from the time
of flurprimidol treatment to the date of the final measurements.
Accumulation of osmotic solutes may have contributed to
the increase in sap osmolality and the decrease in Ψπ(100) in the
nonirrigated flur(+) seedlings (Figures 3B and 3C). Osmotic
adjustment usually develops in plants that are stressed slowly;
however, it is not detectable in all plant species and is less
common in woody plants than in herbaceous plants (Kramer
1983, Kozlowksi et al. 1991). The effects of flurprimidol on
osmoregulation or solute accumulation are not known, and
studies on solute potential and solute accumulation in plants
treated with paclobutrazol, a triazole growth inhibitor, have
yielded conflicting results. Mature leaves of paclobutrazoltreated apple seedlings had lower solute potentials than mature
leaves of untreated controls, but no differences in Ψp were
observed (Swietlik and Miller 1983). Increased concentrations
of non-structural carbohydrates were found in tissues of
paclobutrazol-treated apple seedlings (Wang et al. 1985) and
paclobutrazol-treated apple trees (Wieland and Wample 1985).
However, Vu and Yelenosky (1992) observed lower concentrations of soluble sugars in paclobutrazol-treated sweet orange
plants than in untreated controls.
Flurprimidol treatment reduced A, probably as a result of the
reduction in gs. There are conflicting conclusions about the
effect of flurprimidol and other growth inhibitors on A and gs
depending on whether results are expressed for an entire plant,
normalized on a leaf area basis, or expressed as a ratio in terms
of water use efficiency (Marquard 1985, Wieland and Wample
1985, Davis and Sankhla 1986, Sterrett et al. 1989, Rieger and
Scalabrelli 1990, Steinberg et al. 1991a, Frymire and Hender-
102
PREMACHANDRA, CHANEY AND HOLT
son-Cole 1992, Vu and Yelenosky 1992, Zhou and Xi 1993).
Cathey (1975) observed that ancymidol arrested stomatal
opening, but only at a dosage much higher than that adequate
for growth control. The concentration of flurprimidol applied
in this study (1.0 mM, or 312 mg per pot) is slightly less than
the dose recommended for soil application of a similar growth
inhibitor to white ash trees.
In other studies, PGR treatment reduced E and water use
(Atkinson and Chauhan 1987, Schuch 1994). Total water use
was 33% lower in uniconazole-treated hibiscus plants than in
control plants; however, WUE was lower in treated plants than
in control plants (Steinberg et al. 1991b). We also observed
lower WUE (A per unit of H2O transpired) in flur(+) seedlings
than in flur(−) seedlings at leaf Ψw values in the range of −3 to
−5 MPa (Figure 6A); however, GEE (A per unit of gs) was
greater in flur(+) seedlings than in flur(−) seedlings at leaf Ψw
values less than −2 MPa (Figure 6B). A similar response was
observed in paclobutrazol-treated nectarine trees and was accompanied by increases in Ci and mesophyll conductance
(DeJong and Doyle 1984), suggesting an increase in mesophyll (intracellular) conductance for CO2. In contrast, we
found no significant difference in Ci between flur(+) and flur(−)
seedlings under either irrigated or nonirrigated conditions. The
discrepancy between these results may be associated with the
determination of Ci. In the conventional calculation of Ci, it is
assumed that photosynthesis and transpiration are uniform
throughout a leaf. However, as discussed by Terashima et al.
(1988), it cannot be assumed that the stomatal response is
uniform over the leaf. Furthermore, variation in stomatal aperture could increase as leaf water stress develops.
The decrease in stomatal conductance in response to flurprimidol treatment may be mediated through its effect on ABA
metabolism. Ancymidol, an analog of flurprimidol, promotes
ABA biosynthesis in the fungus Cercospora rosicola and in the
mesocarp tissue of avocado (Norman et al. 1983, Cowan and
Railton 1987). Shortly after treatment with pyrimidine compounds, increases in ABA concentrations have been observed
in cell suspensions, detached leaves, and young plants (Grossmann 1992). In leaves, the increase in ABA concentration is
accompanied by an increase in stomatal resistance and a decrease in transpiration rate, and in suspension cultures of
oilseed rape cells, it is closely correlated with enhanced potassium and water content of the cells (Häuser et al. 1990,
MacKay et al. 1990). These results are similar to the responses
we observed in white ash seedlings treated with flurprimidol.
Flurprimidol treatment improved plant water status of the
nonirrigated seedlings by increasing Ψw, RWC, and Ψp, and
decreasing Ψπ(100) . Although water use efficiency was not
improved by the flurprimidol treatment, plant water use was
reduced as a result of the decline in E; consequently, flur(+)
seedlings survived longer than flur(−) seedlings without watering. Thus, although total photosynthetic production may be
lower in flur(+) seedlings than in flur(−) seedlings, flurprimidol enhances plant survival under water stress conditions. The
greater sap osmolality and lower Ψπ(100) in flur(+) seedlings
than in flur(−) seedlings suggest that flurprimidol improves
plant water status by facilitating osmotic adjustment under
water stress conditions.
Acknowledgments
This research was funded by a grant from DowElanco, Indianapolis,
Indiana, USA. Robert J. Joly provided careful review of the manuscript.
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© 1997 Heron Publishing----Victoria, Canada
Gas exchange and water relations ofFraxinus americana affected by
flurprimidol
GNANASIRI S. PREMACHANDRA, WILLIAM R. CHANEY and HARVEY A. HOLT
Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN 47907, USA
Received August 30, 1995
Summary Effects of flurprimidol on plant water relations
and leaf gas exchange were investigated in one-year-old white
ash (Fraxinus americana L.) seedlings subjected to soil water
deficits. Flurprimidol (20 mg kg −1 of soil equivalent) was
applied to the soil surface of pot-grown seedlings after shoot
growth was completed. Two months after flurprimidol application, water was withheld from one-half of the seedlings. Leaf
water relations and gas exchange parameters were measured 5,
7, 10, 14, 18 and 22 days after withholding water. Under both
irrigated and nonirrigated conditions, flurprimidol treatment
resulted in reduced net CO2 assimilation rate and transpirational water loss of seedlings as a result of decreased stomatal
conductance. Consequently, flurprimidol-treated seedlings had
higher leaf water potential and relative water content than
untreated seedlings. Nonirrigated flurprimidol-treated seedlings also had greater turgor and sap osmolality and lower
osmotic potential at full turgor than seedlings in the other
treatments, indicating that flurprimidol increased osmotic adjustment. Under water-stress conditions, water use efficiency
was lower and gas exchange efficiency was higher in flurprimidol-treated seedlings than in untreated seedlings, suggesting
that flurprimidol treatment enhances survival of plants subjected to soil water deficits.
ery stock with a growth inhibitor may improve plant survival
and quality at times when water is limited.
Flurprimidol (α-(1-methylethyl)-α-[4-(trifluoromethoxy)
phenyl]-5-pyrimidine-methanol), a gibberellin synthesis inhibitor (Rademacher 1991), is used to control shoot growth in
trees. Flurprimidol and other pyrimidine PGRs modify the
growth of plants by causing shorter internodes and smaller
leaves (Davis and Curry 1991). In addition to interfering with
gibberellin biosynthesis, pyrimidine compounds also promote
the biosynthesis of abscisic acid (ABA) (Cowan and Railton
1987, Grossmann 1992). Both abscisic acid and gibberellins
are synthesized via the isoprenoid pathway, and the two plant
hormones often exhibit opposing physiological activities
(Loveys and Milborrow 1984). The role of ABA in stomatal
functioning and plant water relations is well documented (Kozlowski et al. 1991). Although the growth regulating effects of
flurprimidol have been investigated in several herbaceous species, little is known about its effects on the physiological
processes of tree species. Therefore, we have studied the effects of flurprimidol on the plant water relations, leaf gas
exchange, and water use efficiency of irrigated and nonirrigated white ash (Fraxinus americana L.) seedlings to test the
hypothesis that flurprimidol treatment enhances plant tolerance to water stress.
Keywords: osmotic adjustment, plant growth regulators, water
stress.
Materials and methods
Introduction
Several plant growth regulators (PGRs) are used to suppress
shoot growth in horticultural crops and urban trees (Davis and
Curry 1991). In addition to controlling growth, PGRs also
increase root/shoot ratios (Early and Martin 1988, Numbere et
al. 1992), decrease stomatal conductance (Armitage et al.
1984, DeJong and Doyle 1984, Steinberg et al. 1991a), increase root hydraulic conductivity and xylem pressure potential (Vaigro-Wolff and Warmund 1987, Rieger and Scalabrelli
1990) and decrease plant water use by reducing transpiration
(Atkinson and Chauhan 1987, Schuch 1994). Because all of
these effects tend to make plants more drought tolerant and
improve their water use efficiency, Frymire and HendersonCole (1992) suggested that treatment of container-grown nurs-
One-year-old seedlings of white ash were obtained from the
Indiana State Tree Nursery and graded to uniform size. On
April 10, 1994, each of 100 seedlings was planted in a 15.2-liter plastic pot containing a 7/3/2 (v/v/v) mix of top soil/peat
moss/perlite amended with (per m3): 680 g Ca(H2PO4)2, 454 g
KNO3, 454 g MgSO4, 3.6 kg ground limestone and 57 g fritted
trace elements. The seedlings were grown in a greenhouse at
Purdue University, West Lafayette, Indiana, USA, in a natural
photoperiod at a maximum day/minimum night temperature of
approximately 28/23 °C until terminal shoot growth was completed and the leaves were fully expanded.
After leaf growth ceased in early June, one half of the
seedlings were randomly selected for flurprimidol treatment.
On June 14, 1994, flurprimidol was applied to the soil at the
rate of 20 mg kg −1 of soil (312 mg flurprimidol per15.6 kg soil
in each pot). No additional leaf flushes occurred in either
98
PREMACHANDRA, CHANEY AND HOLT
flurprimidol-treated (flur(+)) or untreated (flur(−)) seedlings.
On August 11, 1994, two months after flurprimidol application, one half of the flurprimidol-treated and untreated seedlings were randomly selected for water stress treatment. They
were not watered again for the 22-day duration of the study.
The other half of the treated and untreated seedlings continued
to receive regular watering to resaturation every other day and
served as irrigated controls.
Leaf water relations and gas exchange parameters were
measured to compare flurprimidol-treated and untreated seedlings under irrigated and nonirrigated conditions 5, 7, 10, 14,
18 and 22 days after water was withheld. Before dawn on the
day of measurement, 20 seedlings (five replicates from each of
the four treatments) were moved to a large growth chamber
where sampling could be carried out under conditions of uniform light, temperature and humidity. Light from six 1000-W
high-pressure sodium vapor lamps (Energy Technics, York,
PA) was filtered through a 3-cm layer of water, providing an
incident photosynthetic photon flux to the sample leaves of
approximately 900 µmol m −2 s −1. Temperature and relative
humidity were 25 ± 2 °C and 40 ± 5%, respectively, and
ambient CO2 concentration was 340--360 µl l −1. Plants were
equilibrated under these conditions for 3 h before measurements were initiated.
Measurement of leaf gas exchange
Net CO2 assimilation rate (A), stomatal conductance (gs), transpiration rate (E) and internal CO2 concentration (Ci) were
measured between 1000 and 1200 h on two leaflets of the two
uppermost, fully expanded leaves with an ADC portable photosynthetic and transpiration measurement system (Analytical
Development Company Limited, Hoddesdon, England). Instantaneous water use efficiency (WUE) was calculated as the
ratio of A/E (mmol mol −1). Intrinsic gas exchange efficiency
(GEE) was calculated as the ratio of A/gs (µmol mol −1).
Measurements of leaf water potential, osmotic potential and
relative water content
Discs (0.5-cm diameter) were cut from the same leaflets used
for gas exchange measurement and immediately put in thermocouple psychrometer sample chambers (Wescor C-52; Wescor
Inc., Logan, UT) and allowed to equilibrate for 4 h. Leaf water
potential (Ψw) was then measured psychrometrically with a
microvoltmeter. The Ψw values were accurate to ± 0.05 MPa
and were comparable to Ψw values measured with a pressure
chamber. Afterwards, the leaf discs were frozen and thawed in
the psychrometer sample chambers and their osmotic potential
(Ψπ) was measured psychrometrically. Leaf Ψπ was corrected
for the dilution of symplastic sap by apoplastic water, which
occurs when cell walls are broken during freezing and thawing
of leaf discs (Tyree 1976); a constant apoplastic fraction of
0.16 was assumed (Parker and Pallardy 1987). Turgor (Ψp) was
calculated by subtracting the corrected Ψπ from Ψw. Relative
water content (RWC) was computed as RWC = (FW--DW)/
(TW--DW) (where FW = fresh weight, DW = dry weight and
TW = turgid weight), using 1-cm-diameter discs excised from
leaflets of the same two leaves used for previous measure-
ments. Osmotic potential at full turgor (Ψπ(100) ) was calculated
as Ψπ(100) = Ψπ (RWC − AWC)/(1.0 − AWC), where AWC is the
apoplastic water content (Wilson et al. 1979). Osmotic adjustment (OA) was estimated as the difference between the Ψπ(100)
values estimated in leaves of irrigated and nonirrigated plants.
Measurement of sap osmolality
Several leaflets from the two leaves that were used for gas
exchange and water relations measurements were frozen in
sealed polyethylene freezer bags. Leaf samples were thawed
and centrifuged at 1000 g for 20 min at 6--8 °C to extract cell
sap. Sap osmolality was measured with a Wescor Model
5100C vapor pressure osmometer (Wescor Inc.) and was corrected for dilution of symplastic sap by apoplastic water.
Statistical analyis
Data were subjected to analysis of variance. For the first four
measurement periods, a 4 × 2 × 2 × 5 factorial design was used,
including four sampling periods, two flurprimidol treatments,
and two watering treatments with five replications. Because
many of the nonirrigated flur(−) seedlings died between sampling days 14 and 18, no measurements were made of seedlings in this treatment for sampling days 18 and 22. Data for
flur(+), nonirrigated seedlings were analyzed in a 6 × 5 completely randomized block design with six sampling periods
and five replications. The least significant difference (LSD) at
the 0.05 level was calculated to determine differences among
means. Regression analyses of the various parameters measured as a function of leaf Ψw were performed and best fit curves
plotted.
Results
Figures 1 and 2 illustrate changes in leaf Ψw, RWC, Ψp, Ψπ(100),
and OA of flurprimidol-treated (flur(+)) and untreated (flur(−))
white ash seedlings under irrigated and nonirrigated conditions
over time. For the first 14 days after withholding water, leaf Ψw
was higher in flur(+) seedlings than in flur(−) seedlings under
both irrigated and nonirrigated conditions (Figure 1A). Leaf
Ψw of irrigated seedlings was 0.21 to 0.36 MPa higher in the
flur(+) treatment than in the flur(−) treatment during this
period. In the nonirrigated treatments, Ψw decreased more
rapidly in flur(−) seedlings than in flur(+) seedlings (−5.09
versus −2.13 MPa on Day 14) (Figure 1A). Leaf Ψw of nonirrigated flur(+) seedlings reached −3.43 and −5.00 MPa 18 and
22 days after withholding water, respectively. Leaves for
which a Ψw value of −5 MPa was measured were not severely
wilted, presumably because of their high cellulose content, but
the leaf margins and small areas of the leaves were beginning
to turn brown.
Relative water content in nonirrigated flur(−) seedlings decreased rapidly after water was withheld, declining from 81.5
to 53.6% between Days 5 and 14. The corresponding RWC
values for nonirrigated flur(+) seedlings were 87.7 to 83.7%.
With further exposure to water stress, RWC in nonirrigated
flur(+) seedlings decreased to 63.5 and 43.1% by Days 18 and
22, respectively (Figure 1B).
FLURPRIMIDOL IMPROVES TOLERANCE TO WATER STRESS
Under both irrigated and nonirrigated conditions, turgor was
significantly greater in flur(+) seedlings than in flur(−) seedlings, except in irrigated seedlings on Days 7 and 14 (Figure
2A). Irrigated flur(+) seedlings exhibited a Ψp of 2.01 to 2.25
MPa compared with 1.69 to 2.13 MPa for irrigated flur(−)
seedlings. Turgor of nonirrigated flur(−) seedlings decreased
with time and reached zero at Day 14. Nonirrigated flur(+)
seedlings maintained higher Ψp at lower leaf Ψw and for a
longer time than flur(−) seedlings, exhibiting a Ψp of 0.4 MPa
even after water was withheld for 22 days (Figures 2A and 3A).
In both flur(−) and flur(+) seedlings, withholding water for
14 days resulted in a significant decrease in Ψπ(100) (Figure 2B). Osmotic potential at full turgor of nonirrigated flur(+)
seedlings decreased by 0.6 MPa from Days 5 to 18. At Day 22,
Ψπ(100) of nonirrigated flur(+) seedlings was 0.2 MPa above
that at Day 5 (Figure 2B). Nonirrigated flur(+) seedlings maintained a lower Ψπ(100) at a given leaf Ψw than flur(−) seedlings,
except at Ψw values below −5 MPa (Figure 3B).
In the nonirrigated treatments, osmotic adjustment was evident in both flur(−) and flur(+) seedlings (Figure 2C). The
greater osmotic adjustment in flur(−) seedlings than in flur(+)
seedlings at 10 and 14 days after withholding water was a
result of exposure of flur(−) seedlings to a higher degree of
water stress as measured by Ψw. Sap osmolality was greater in
flur(+) seedlings than in flur(−) seedlings over a wide range of
leaf Ψw values (Figure 3C).
The effects of irrigation treatment on A and gs were observed
as soon as 5 days after withholding water in flur(−) seedlings;
however, in flur(+) seedlings, the effects were observed only
during the later stages of stress development (Figures 4A and
4B). For example, in flur(+) seedlings, there were no differ-
Figure 1. Leaf water potential (A) and relative water content (B) of
flurprimidol-treated (Flur+) and untreated (Flur−) white ash seedlings
grown in irrigated (Irri) or nonirrigated (Non) conditions at 5, 7, 10,
14, 18 and 22 days after withholding water. The LSD is at 0.05 level.
99
ences in A and gs between nonirrigated and irrigated seedlings
until Days 14 and 10, respectively. Net CO2 assimilation in
irrigated flur(+) seedlings was about 50% of that in irrigated
flur(−) seedlings. Similarly, gs was lower in irrigated flur(+)
seedlings than in irrigated flur(−) seedlings. In flur(−) seedlings, both A and gs decreased rapidly with increasing water
stress and reached values that were 87 and 89% lower on Days
5 and 14, respectively, than in irrigated flur(−) seedlings. Net
CO2 assimilation rate in nonirrigated flur(+) seedlings declined by 93%, but only after withholding water for 22 days.
Likewise, stomatal conductance in nonirrigated flur(+) seedlings declined by 95% between Days 5 and 22. Flur(+) seedlings exhibited lower A and gs than flur(−) seedlings over a
wide range of leaf Ψw values (Figures 5A and 5B).
Transpiration rate was lower in nonirrigated than in irrigated
seedlings in both flur(+) and flur(−) treatments at all sampling
periods up to 14 days after withholding water (Figure 4C).
Flurprimidol reduced E by 45% in irrigated seedlings. In
nonirrigated seedlings, flur(+) seedlings exhibited a lower E
than flur(−) seedlings up to 7 days after withholding water, and
thereafter, flur(+) seedlings exhibited a higher E than flur(−)
seedlings. Transpiration rates of nonirrigated flur(+) seedlings
on Days 18 and 22 were approximately 53 and 12%, respectively, of those on Day 5. Nonirrigated flur(+) seedlings had a
lower E than flur(−) seedlings over a wide range of leaf Ψw
Figure 2. Turgor (A), osmotic potential at full turgor (B) and osmotic
adjustment (C) of flurprimidol treated (Flur+) and untreated (Flur−)
white ash seedlings grown in irrigated (Irri) or nonirrigated (Non)
conditions at 5, 7, 10, 14, 18 and 22 days after withholding water. The
LSD is at 0.05 level.
100
PREMACHANDRA, CHANEY AND HOLT
Figure 3. Regression of turgor (A), osmotic potential at full turgor (B)
and sap osmolality (C) as functions of leaf water potential in flurprimidol-treated (Flur+) and untreated (Flur−) white ash seedlings. Variables were measured in leaves of container-grown seedlings during a
22-day period in which water was withheld. The Flur+ and Flur−
treatment effects were significantly different (P = 0.05).
values (Figure 5C). Neither the flurprimidol treatments nor the
irrgation treatments affected Ci (Figure 4D).
The effects of a range of leaf Ψw values on water use
efficiency and gas exchange efficiency of flur(+) and flur(−)
white ash seedlings are shown in Figure 6. Water use efficiency
of flur(+) seedlings was lower than that of flur(−) seedlings at
leaf Ψw values ≤ −2 MPa, except at Ψw = −5 MPa, where they
were similar. Flur(+) seedlings had a higher WUE than flur(−)
seedlings at high leaf Ψw (−1 MPa). In contrast, GEE was
significantly greater in flur(+) seedlings than in flur(−) seedlings only when leaf Ψw was less than −2 MPa.
Discussion
Flurprimidol decreased stomatal conductance in white ash
seedlings even when soil water was not limiting. On all measurement days, stomatal conductance in irrigated flur(+) seedlings was lower than in irrigated flur(−) seedlings, resulting in
lower A and E. In the nonirrigated treatments, gs of flur(+)
seedlings was only reduced below that of flur(−) seedlings
Figure 4. Net CO2 assimilation rate (A), stomatal conductance (B),
transpiration rate (C) and internal CO 2 concentration (D) of flurprimidol-treated (Flur+) and untreated (Flur−) white ash seedlings grown in
irrigated (Irri) or nonirrigated (Non) conditions for 5, 7, 10, 14, 18 and
22 days after withholding water. The LSD is at 0.05 level.
14 days after withholding water. Nonirrigated flur(+) seedlings had higher Ψw, Ψp, and leaf RWC than nonirrigated
flur(−) seedlings, and only the flur(+) seedlings survived for
more than 14 days after water was withheld, presumably as a
result of reduced E, reduced plant water use, and increased
turgor maintenance. Osmotic adjustment occurred in flur(−)
seedlings on Days 10 and 14, and occurred in flur(+) seedlings
on Day 14, as indicated by decreased Ψπ(100) and increased sap
osmolality.
The values of Ψw (−5 MPa) determined for leaves of nonirrigated seedlings are low compared with published values. The
leaves of most vascular plants are irreversibly damaged at such
low water potentials as a result of mechanical tearing of protoplasm and degradation of membranes under the extreme tensions developed within cells (Gaff 1980). For example, Cleary
and Zaerr (1980) reported that vigor of ponderosa pine seedlings declined progressively between Ψw values of −2 and
−5 MPa, and the seedlings died when Ψw reached −5 MPa.
FLURPRIMIDOL IMPROVES TOLERANCE TO WATER STRESS
101
Figure 6. Regression of water-use efficiency (A) and intrinsic gas-exchange efficiency (B) as functions of leaf water potential in flurprimidol-treated (Flur+) and untreated (Flur−) white ash seedlings during a
22-day period in which water was withheld. Water-use efficiency was
calculated as the ratio of net CO2 assimilation rate/transpiration rate,
and intrinsic gas-exchange efficiency was calculated as the ratio of net
CO2 assimilation rate/stomatal conductance. The Flur+ and Flur−
treatment effects were significantly different (P = 0.05).
Figure 5. Regression of net CO2 assimilation rate (A), stomatal conductance (B) and transpiration rate (C) as functions of leaf water
potential in flurprimidol-treated (Flur+) and untreated (Flur−) white
ash seedlings. Variables were measured in leaves of container-grown
seedlings during a 22-day period in which water was withheld. The
Flur+ and Flur− treatment effects were significantly different
(P = 0.05).
There are few studies on the effects of flurprimidol or other
pyrimidine growth inhibitors on plant water relations and gas
exchange. Pyrimidine growth inhibitors reduce water use
(Johnson 1974, Barrett and Nell 1981), and the reduction is
partially related to the smaller leaf area of the treated plants
(Davis et al. 1986, Vaigro-Wolff and Warmund 1987, Sterrett
et al. 1989). Barrett (1983) reported a reduction of 35 and 39%
in total plant transpiration in poinsettias treated with EL-500
(flurprimidol) and ancymidol, respectively, whereas on a per
leaf basis the difference in transpiration was only 12% for both
compounds.
High root/shoot ratios are normally observed in PGRtreated plants (Early and Martin 1988, Numbere et al. 1992),
and could lead to improved water absorption and water availability to shoots (Steinberg et al. 1991b). However, in the
present study, the flurprimidol treatment was applied after
terminal shoot growth was completed and leaves were fully
matured. Hence, there was no new shoot growth from the time
of flurprimidol treatment to the date of the final measurements.
Accumulation of osmotic solutes may have contributed to
the increase in sap osmolality and the decrease in Ψπ(100) in the
nonirrigated flur(+) seedlings (Figures 3B and 3C). Osmotic
adjustment usually develops in plants that are stressed slowly;
however, it is not detectable in all plant species and is less
common in woody plants than in herbaceous plants (Kramer
1983, Kozlowksi et al. 1991). The effects of flurprimidol on
osmoregulation or solute accumulation are not known, and
studies on solute potential and solute accumulation in plants
treated with paclobutrazol, a triazole growth inhibitor, have
yielded conflicting results. Mature leaves of paclobutrazoltreated apple seedlings had lower solute potentials than mature
leaves of untreated controls, but no differences in Ψp were
observed (Swietlik and Miller 1983). Increased concentrations
of non-structural carbohydrates were found in tissues of
paclobutrazol-treated apple seedlings (Wang et al. 1985) and
paclobutrazol-treated apple trees (Wieland and Wample 1985).
However, Vu and Yelenosky (1992) observed lower concentrations of soluble sugars in paclobutrazol-treated sweet orange
plants than in untreated controls.
Flurprimidol treatment reduced A, probably as a result of the
reduction in gs. There are conflicting conclusions about the
effect of flurprimidol and other growth inhibitors on A and gs
depending on whether results are expressed for an entire plant,
normalized on a leaf area basis, or expressed as a ratio in terms
of water use efficiency (Marquard 1985, Wieland and Wample
1985, Davis and Sankhla 1986, Sterrett et al. 1989, Rieger and
Scalabrelli 1990, Steinberg et al. 1991a, Frymire and Hender-
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PREMACHANDRA, CHANEY AND HOLT
son-Cole 1992, Vu and Yelenosky 1992, Zhou and Xi 1993).
Cathey (1975) observed that ancymidol arrested stomatal
opening, but only at a dosage much higher than that adequate
for growth control. The concentration of flurprimidol applied
in this study (1.0 mM, or 312 mg per pot) is slightly less than
the dose recommended for soil application of a similar growth
inhibitor to white ash trees.
In other studies, PGR treatment reduced E and water use
(Atkinson and Chauhan 1987, Schuch 1994). Total water use
was 33% lower in uniconazole-treated hibiscus plants than in
control plants; however, WUE was lower in treated plants than
in control plants (Steinberg et al. 1991b). We also observed
lower WUE (A per unit of H2O transpired) in flur(+) seedlings
than in flur(−) seedlings at leaf Ψw values in the range of −3 to
−5 MPa (Figure 6A); however, GEE (A per unit of gs) was
greater in flur(+) seedlings than in flur(−) seedlings at leaf Ψw
values less than −2 MPa (Figure 6B). A similar response was
observed in paclobutrazol-treated nectarine trees and was accompanied by increases in Ci and mesophyll conductance
(DeJong and Doyle 1984), suggesting an increase in mesophyll (intracellular) conductance for CO2. In contrast, we
found no significant difference in Ci between flur(+) and flur(−)
seedlings under either irrigated or nonirrigated conditions. The
discrepancy between these results may be associated with the
determination of Ci. In the conventional calculation of Ci, it is
assumed that photosynthesis and transpiration are uniform
throughout a leaf. However, as discussed by Terashima et al.
(1988), it cannot be assumed that the stomatal response is
uniform over the leaf. Furthermore, variation in stomatal aperture could increase as leaf water stress develops.
The decrease in stomatal conductance in response to flurprimidol treatment may be mediated through its effect on ABA
metabolism. Ancymidol, an analog of flurprimidol, promotes
ABA biosynthesis in the fungus Cercospora rosicola and in the
mesocarp tissue of avocado (Norman et al. 1983, Cowan and
Railton 1987). Shortly after treatment with pyrimidine compounds, increases in ABA concentrations have been observed
in cell suspensions, detached leaves, and young plants (Grossmann 1992). In leaves, the increase in ABA concentration is
accompanied by an increase in stomatal resistance and a decrease in transpiration rate, and in suspension cultures of
oilseed rape cells, it is closely correlated with enhanced potassium and water content of the cells (Häuser et al. 1990,
MacKay et al. 1990). These results are similar to the responses
we observed in white ash seedlings treated with flurprimidol.
Flurprimidol treatment improved plant water status of the
nonirrigated seedlings by increasing Ψw, RWC, and Ψp, and
decreasing Ψπ(100) . Although water use efficiency was not
improved by the flurprimidol treatment, plant water use was
reduced as a result of the decline in E; consequently, flur(+)
seedlings survived longer than flur(−) seedlings without watering. Thus, although total photosynthetic production may be
lower in flur(+) seedlings than in flur(−) seedlings, flurprimidol enhances plant survival under water stress conditions. The
greater sap osmolality and lower Ψπ(100) in flur(+) seedlings
than in flur(−) seedlings suggest that flurprimidol improves
plant water status by facilitating osmotic adjustment under
water stress conditions.
Acknowledgments
This research was funded by a grant from DowElanco, Indianapolis,
Indiana, USA. Robert J. Joly provided careful review of the manuscript.
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