Changes in Nitrate Accumulation and Growth of Endive Plants

JOURNAL OF PLANTNUTRTIlON, 20(10),1255-1266 (1997)
Changes in Nitrate Accumulation and
Growth of Endive Plants During Light
Period as Affected by Nitrogen level
and Formi
P. Santamaria,. A. Elia,b and M. Gonnellab
a/nst;tute 01 Agronomy and F;e/d Crops, Un;vers;ty 01 Bar;, Bar; 70/26, /ta/y
b/nstitute olVegetab/e Cropslor Process;ng, CNR. Bar; 70126, /ta/y
ABSTRACT
Endive plants (C;choriumendivia L. varocrispum Hegi) were bydroponicatly
grown in a growth chamber to asse55, 7 and 14 days after beginning of
treatments (DAT), the variation ofleafnitrate (NO) accumulationand growth
during the Iight period in relation to both nitrogen (N) availability (8 and 16
mM) and form percentage ratio [50:50 and O:100 ammonium (NH4):NO)]in
the nutrient solution. 80th 7 and 14 DAT, fresh weight and leaf area linearly
increased during the lig~t period without influence of N treatments. Dry
matter (d.m.) percentage increased over the 12 hours of Iight. Leaf NO)
concentration decreased during the Iight period more with 50:50 than with
O:100 NH4:NO) ratio: 7 DAT NO) decreased by 25% with the former N
treatment and only by 4% with the latter. After 7 days more, the difference
between the two N form ratios was more evident: NO) decreased by 12%
with the mixed N fonn, while increased by 10% with 100% NO)-N in the
IThe work was carried out at tbe Institute ofVegetable Crops for Processing oftbe Italian
National Research Council (CNR), Director Prof. Vito V. Bianco.
1255
1256
SANT AMARIA, EllA, AND GONNELLA
nutrient solution. Nitrate leafconcentrationwashigherwith 16mM compared
to 8 mM ofNO3-N (11.3 and 8.5 g/100 g ofd.m., respectively, 14 DAT).
Total N leaf content was higher with 16 than with 8 mM of NO3-N in the
nutrient solution and was not influencedby the two N form ratios. Chloride
(Cl) leaf concentration was higher with 8 that with 16 mM of N and more
with the 50:50 than 0:100 NH.:NO3ratio.
INTRODUCTION
Leafy vegetables accumulate nitrates especially when grown in high NO3-N
availability and low radiation. Nitrates are compounds potentially toxic for human
health (Walker, 1990). In plant tissues, NO3 concentration is not constant
throughout the day; it increases during the night and decreases during the light
period (Minotti and Stankey, 1973; Steingrover et al., 1986; Le Bot and Kirkby,
1992). .. Endive(CichoriumendiviaL. varocrispumHegi)is a leafyvegetable
present on fresh produce markets ali year. Schonbeck et al. (1991) reported that
endive plants harvested in March accumulate nitrates from 52 to 68% less than
plants harvested in November. Some studies report relatively low levels ofnitrate
in endive compared to other nitrate accumulating vegetables, such as lettuce,
spinach, and celery (Roorda van Eysinga and Spaans, 1986; Vegter and Tanis,
1990). However, in experiments by Schonbeck et al. (1991) and Reinink et al.
(1994) the level of nitrate in endive was similar to the levels found in lettuce
cultivars. Reinink et al. (1994), that reported genetic variations in the nitrate
content between cultivars of endive, found that there is only a low possibility of
reducing the nitrate content of endive by cultivar choice.
In our previous work in solution culture, by supplying endive plants with mixed
form nitrogen, nitrogen yield efficiency increased while leaf nitrate content
decreased as compared to nitrate-fed plants (Santamaria et al., 1995; Santamaria
and Elia, 1997). The aim ofthe present work was to evaluate the influence oftwo
levels of N, administered in mixed ammonium-nitrate or in nitrate form on the
variation of nitrate accumulation and growth of endive plants during the light
periodo
MATERIALS AND METHODS
Endive seedlings, grown in a cellular tray till5th true leaf stage, were transferred
after rinsing of roots with tap water to rectangular prismatic plastic pots (60 cm
long, 17 cm large, 13 cm wide) suitably covered to avoid evaporation of the
nutrient solution. Plants were grown for two weeks in a Conviron Model PGW36
growth chamber under the following conditions: day temperature 20°C, night
temperature 16°C, relative humidity 75%, photoperiod 12 h, iIIumination supplied
by Sylvania (Canada) cool white high intensity fluorescent lamps supplemented
with 50 W incandescent lamps [irradianceat the leaf surface 300 flmoV(m2s)].
NITRA TE ACCUMULA TION AND GROWTH OF ENDIVE PLANTS
TABLE I.
Treatments
NH,-N:NO)-N
(mM)
4:4
0:8
8:8
O: 16
Composition of nutrient solutions for differenttreatments.'
Compound and concentration (mM)
Ca(NO])2
O
4
O
4
'The other micronutrients
Amon
1257
NaNO]
NH,NO)
CaCI2
MgSO,
K2HPO,.
O
O
O
8
4
O
4
O
2
O
2
O
l
I
l
I
O
2
O
2
were the SarTIe for ali treatments
according
K2SO, Ca(H2PO,)2
3
1
3
I
2
O
2
O
lo Hoagland
and
(1950).
Four N treatments were studied based on the combination of two N levels (8
and 16 mM) and two molar NH.:NO] percentage ratios (50:50 and 0:100) in a
randomized complete block design with three replications (pots with 15 seedlings).
Compounds used to create the different N-ratio and N-rate treatments are reported
in Table I.
Every day, in each pot distilled water was added to make up for the volume of
solution, and NaOH (2N) or HzSO. (I N) were added to keep pH in the range of 56. The nutrient solution, replaced weekly, was continuously bubbled through
pots to supply oxygen.
Seven days after plants had been moved in the nutrient solution, starting from
7.00 (beginning of light peri od) to 19.00 (beginning of dark period), two plants
from each pot were sampled at three hour intervals. The above procedure was
repeated after one week, but only one plant per pot was sampled (an error in
preparation of nutrient solution of the treatment with 16 mM of N in the 50:50
NH.:NO] ratio do not allow to discuss related results).
Fresh weight and leaf area (measured by LI-3100 leaf area meter; LI-COR,
Lincoln, NE) in both samplings were evaluated. The material was dried in a
thermoventilated oven at 65°C until constant weight was reached. The material
was then weighed, finely ground, and used for the quantitative chemical analyses
of NO) and CI, determined by ion chromatography (Elia et al., 1996), and total
Kjeldahl N (only in the last sampling) by Kjeltec Auto 1030 Analyzer.
Data were subjected to SAS's (SAS Institute, Cary, N.C.) generallinear model
procedure, considering separately the 1\'10sampling times (Tables 2 and 3). A
"split-block" experimental scheme was adopted since the t\vo main factors (hours
of sampling during the light period: "LlGHT' and "N") were in strips orthogonal
bet\veen them. For the "LIGHT' factor the relation with the response variables
was assessed by partitioning the sums of the squares into components that were
associated with linear, quadratic, and residual terms (Steel and Torrie, 1980).
1258
SANTAMARIA, ELlA, AND GONNELLA
TABLE 2. Analysls ofvariance for freshmatter (A), leaf area(B), dry matter (C), nitrate
(D), and chloride(E) of endiveleaf atfiveday-lighttimes and grown withfourN treatments
for one week.
A
Sourceof variation
df
LlGHT
4
B
C'
IY
SignificanceofF test
... ..
light (linear)
light (quadratic)
1
I
ns
ns
light(residua!)
Errar l
2
ns
ns
8
N
3
ns
N rate
1
1
1
6
ns
ns
ns
ns
NH.:NOJratio
N rate x NH.:NOJratio
Errar2
LlGHTx N
LIGHTx N RATE
light (!iDear) x N rate
light (quadratic) x N rate
light (residua!) x N rate
LlGHT x NH.:NO) RATIO
light (linear) x NH.:NO) rado
light (residua!) x NH.:NO) ratio
LlGHT x N RATE x NH.:NO] RATIO
Errar 3
12
4
1
1
2
4
1
3
4
24
... .....
...
...
E"
..
..
..
ns
..
ns
ns
ns
...
ns
..
...
ns
ns
ns
ns
...
ns
ns
ns
ns
ns
ns
DS
ns
ns
..
ns
DS
ns
DS
DS
DS
ns
DS
DS
DS
DS
DS
ns
DS
DS
DS
ns
DS
ns
ns
ns
ns
ns
ns
ns
ns
DS
ns
DS
ns
ns
ns
ns
...
DS
.
...
ns
aANOV A was performed on square root-transformed data.
ns, " .', .. .Nonsignificant or significantat P<0.05,0.01, or 0.001, respectively.
RESULTS
Growth
Both 7 and 14 days DAT, fresh weight and leaf area linearly increased during
the light period (Figures 1 and 2). These pattems were not significantly affected
by the N treatments (Tables2 and 3), even if 14DAT freshweight'increasedmore
with 50:50 NH.:NO] ratio than with 100% ofNO3-N (P=O.0511).
In the first sampling, the percentage of leaf dry matter (d.m.) progressively
rose after the first three hoUÌ'Sof light and reached 7.4 glI00 g of fresh matter
(f.m.). Instead 14 DAT, dry matter percentageincreased linearlyby 5% over tbe
12 hours of light (Figure 3). At botb samplings,higher dry matter percentages
with 100% of NO]-N than with the ammonium-nitratesolution were recorded
(Table 4). A similar pattem was shown by the root dry matter (data not shown).
NITRATEACCUMULATION AND GROWTHOF ENDIVEPLANTS
1259
TABLE 3. Analysis of variance for fresh matter (A), leaf area (B), dry
matter (C), nitrate (D), and chloride (E) of endive leaf at fiveday-lighttimes
and grown with three N treatments for two weeks.
A
Sourceofvariation
LIGHT
df
4
light(linear)
light(quadratic)
light(residual)
l
l
l
Error l
8
N
2
B
C'
D'
E'
SignificanceofF test
.
.
.. ns.
ils
..
ns
ns
.
ns
ns
ns
ns
.
ns
ns
.
ns
.
ns
.
ns
..
...
N rate
l
ns
ns
ns
NH.:NO) ratio
l
ns
ns
..
.. ...
.*
***
E"or 2
4
LIGlITx N
8
4
4
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
*
*
*.
light x N rate
light x NH.:NO) ratio
l
light (linear) x NH.:NO) ratio
Error 3
16
ns
*.*
.**
'ANOVA was performed on square root-transformeddata.
ns, *, .., ".Nonsignificant or significant at P<0.05, 0.01, or 0.001,
respectively.
C
.!!!20
~ 16
~
DAT
j
E
Q')
.a; 12
~
-£i
8
(;j
4
~
~ cfç
O
-
1
3
Hours
7
---
14
+
L------ ì
6
9
12
of light (n.)
FIGURE l. Leaffresh weight variation in endive plants during the light peri od 7 and 14
days after the beginning oftreatrnent (DA T). V erticallines indicate :!:SE.
1260
SANT AMARIA, EllA, AND GONNELLA
1::- 5001
CtI
DAT -
a.
::- 400
E
1
£.
7 H-
14
I
i
300
---------
1
1
CtI
2!
200
CtI
(;j 100
QJ
-J
o
o
369
12
Hours of light (n.)
FIGURE 2. Leaf area variation in'endive plants during the light period 7 and 14 days
after the beginning oftreatrnent.(DAT). Verticallines indicate:!:SE.
,.
.5
DAT
-
7
---
14
018
o
o
~7
E
-ci 611-:::
,-=------
(;j
Q)
-Jet
O
369
12
Hours of light (n.)
FIGURE 3. Leaf dry matter (d. m.) variation [on fresh matter (f. m.) basis] in endive
plants during the light period 7 and 14 days after the begiMing oftreatrnent (DAT). Vertical
lines indicate :!:SE.
Nitrate and Chloride Variation
Leaf NO) concentration decreased during tbe light period more witb 50:50
tban witb 0:100 NH4:NO3 ratio. Seven DAT, NO) decreased by 24.6% witb tbe
former N treatment and only by.4% witb tbe latter (Figure 4). After 7 days more
tbe difference between tbe two.N treatments was more evident: NO) decreased
by 11.5% with tbe mixed N form and increased by 10.1% witb 100% ofNO3-N in
tbe nutrient solution (Figure 5). Tbe NO) leaf concentration was (on average)
higher witb 16 compared to 8 mM ofN in tbe solution (Table 4).
NITRATE ACCUMULATION
AND GROWTH OF ENDIVE PLANTS
1261
TABLE 4. DI)' matter (d.m.), nitrates (NO), and chloride (Cl) leaf
contents in endive plants 7 (A) and 14 (B) days after beginning ofN
treatments (significances ofmean separation are reported in Tables 2
and 3).
roM
d.m.
A
B
gllOOg f.m.
NO)
A
B
---glI
4:4
0:8
8:8
O: 16
6.3
6.9
6.6
7.0
4.9
5.3
5.8
6.0
NH.-N:NO)"N
5.6
6.6
6.5
CI
A
B
00 g d.m.-------
6.0
8.5
3.6
2.0
2.9
1.5
11.3
3.2
0.8
0.6
The Clleaf concentration was higher with 8 than with 16 mM ofN and more
with the 50:50 than O:100 NH4:NO)ratio (Table 4). Fourteen DAT and by
supplying plants with 8 mM of N, leaf CI concentrationduring the light period
decreasedwith 100% ofNO)-N and increasedwith 50:50NH4:NO)ratio (Figure
6).
Total Nitrogen
At the last sampling, significant differences were detected in total N leaf content
produced by the two N levels (5.1 vs. 6. ì glI00 g of d.m. with 8 and 16 mM ofN,
respectively), but not by the two N form ratios supplied to the nutrient solution
(omitted table).
NH. :NO,
-
0:100
...
50:50
~7
'O
O)
6~'
8
~5
04
Z
01;::
O
369
12
Hours of light (n.)
FIGURE 4. Leafnitrate concentration (d.m. basis) in endive plants during the light period
as affected by N forro ratio in the nutrient solution, after 7 days of treatment. Verticallines
indicate :f:SE.
1262
SANT AMARIA, EllA, AND GONNELLA
NH.:NO,
0:100
...
50:50
I
?9
-=i
801
-
i
7
~
~
---o
'I
1
.
'_"
1
-"
'~'--'-'-
1
05
z
1
0"(
o
369
12
Hours of light (n.)
FIGURE 5. Leafnitrate concentration (d.m. basis) in endive plants during the light period
as affected by N form ratio in the nutrient solution, after 14 days of treatment. Vertical
lines indicate :!:SE.
DISCUSSION AND CONCLUSIONS
The interaction between NH~ and NOJ is complex. The presence ofNH~ in a
nutrient solution may inhibit (Haynes and Goh, 1978; Sloom and Finazzo, 1986;
Lee and Drew, 1989), have little effect (Marcus- Wyner, 1983) or slightly stimulate
(Bloom and Finazzo, 1986) net NOJ uptake (defined as the difference between
influx and effiux). Some authors have also reported interaction between N fonn
and rates (Allen et al., 1985; Allen and Smith, 1986).
NH.:NO,
,. I
-
0:100
--- 50:50 --_.1
. -.' --..t. -'
E 3 t. .' - - - . . . . -- - - - -.
-=i .01
8
.t::.
01
2
-1
U
o
o
369
12
Hours of light (n.)
FIGURE 6. Leaf chloride concentration (d.m. basis) in endive p1ants during the light
period as affected by N form ratio in the nutrient solution, after 14 days of treatrnent.
Verticallines indicate:!:SE.
NITRA TE ACCUMULA nON AND GROWTH OF ENDIVE PLANTS
1263
In the present study, after 14 days of treatment, while no influence was produced
both by the N leve I or by the N form ratio on plant growth, NO) concentration
rose by 33% with increasing N level from 8 to 16 mM and by 42% when changing
from the 50:50 to 0:100 NH.:NO) ratio (Table 4). Total N, instead, was not
influenced by N form ratio, but was higher with the higher N leveL Therefore,
reduced N (calculated as the residual component ofmean total N niinus NO)-N of
the composite sample) was higher when N was supplied in mixed than in nitrate
form (75 and 62% oftotal N, respectively).
The lower nitrate concentrations in plants treated with 8 mM N would suggest
that a great part ofNO) was assimilated in the metabolic pool, whereas little would
have remained available to be accumulated in the reserve pool or in celI vacuoles
as osmotic regulator. In plants grown with 16 mM ofN, instead, the higher NO)
concentration in leaves indicates a greater part of NO) was accumulated in the
reserve pool (Steingrover et aL, 1986; Izmailov et aL, 1992). With an increasing
supply ofNO)-N, the capacity for NO) reduction in the roots becomes a limiting
factor and an increasing proportion of the total N is translocated to the shoots in
the form ofNO) (Marschner, 1996).
When NH. and NO) were supplied together, NH. improved N yield efficiency,
as we had observed in previous research when the yield increase per unit of N
contained in endive heads was higher with the 50:50 than O:100 NH.:NO) ratio
(Santamaria et al., 1995). However, the use of mixed N form reduced leaf dry
matter percentage compared to the 0:100 NH.:NO) ratio (Table 4), suggesting
that the effects ofthe two N forms on growth could partialIy originate from celi
osmotic regulation and expansion, especialIy if NO) is absorbed in excess of
assimilation needs.
Leaf NO) concentration changed during the light period thus confmning that
light has a crucial role in the regulation of the NO) accumulation, because it is
involved in photosynthesis as welI as in uptake, translocation and reduction of
NO) (Mengel and Viro, 1978; Merlo etaL, 1994). AlI theseprocesses are subjected
to diurnal variations (Steingrover et al., 1986; Le Bot and Kirkby, 1992; Dellion
et aL, 1995).
However, only with the 50:50 NH.:NO) ratio, leafNO) concentration decreased
linearly and markedly during light period (Figures 4 and 5). Therefore, NH. may
be assumed to have stimulated NR activity, probably by favoring the new formation
of cytoplasmatic NR ('superinduction' according to Mehrer and Mo~, 1989).
The inductive effect of NH. on NR appearance was attributed quantitatively to
the induction of specific isoforms ofNR by NH. (Schuster et aL, 1989).
During the light period and with nitrate form, leafNO) concentration decreased
slightly (7 DAT, Figure 4) or increased (14 DA T, Figure 5). In the latter case NO)
uptake exceed the reduction.
Some authors consider this to be a luxury
consumption (Salsac et aL, 1987; Koch et aL, 1988).
At both sampling times, a highly significant and inverse correlation emerged
between CI and NO) (r=-0.74). NH. uptake increased CI absorption (Table 4 and
1264
SANT AMARIA, ELlA, AND GONNELLA
Figure 6) to replaee organie anions and NO) and to allow the restoration of the
anion leve l needed to keep the osmotie potenti al at optimal values for growth
(Blom-Zandstra and Lampe, 1983; Soltani et al., 1989).
Light also indueed the linear inerease of fresh weight, dry mass pereentage,
and leaf area (Figures 1,2, and 3). Sprent and Thomas (1984) suggested that
inereases in leaf area by high N may be limited to species that transport most of
their nitrate direetly to the shoot, where it ean be used as part ofthe osmotie foree
driving celi expansion before being used for growth.
In eonclusion, data from this researeh indicate that by supplying N in mixed
form, NO) eoneentration of endive leaves linearly deereases during the light period.
When N is supplied in the nitrate form andlor it is over supplied, biomass yield
does not inerease, but a luxury eonsumption is obtained; in faet, leaf NO)
eoneentration inereases, even during the light periodo
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'è,