Annals of Botany 82 : 389–392, 1998
Article No. bo980683
SHORT COMMUNICATION
Effect of Irradiance on Chlorophyll Estimation with the Minolta SPAD-502 Leaf
Chlorophyll Meter
B E R N T O L A V H O E L* and K N U T A S B J Ø R N S O L H A UG†
* The Norwegian Crop Research Institute, ApelsŠoll Research Centre, N-2858 Kapp, Norway and † Department of
Biology and Nature ConserŠation, Agricultural UniŠersity of Norway, P.O. Box 5014, N-1432 Ac s, Norway
Received : 23 March 1998
Accepted : 12 May 1998
Leaf chlorophyll content may be used as an indirect indicator of crop nitrogen status. Chlorophyll meter values
(SPAD values) taken with the Minolta SPAD-502 chlorophyll meter in the shade plant Oxalis acetosella L. and in
winter wheat (Triticum aestiŠum L.) varied by 15 and 8 %, respectively, with variation in irradiance. The lowest
SPAD-values were measured at high irradiance. During a natural night-day-night cycle SPAD values for winter wheat
were lowest in the middle of the day, highest at low irradiance at dusk and dawn and intermediate in darkness before
dawn and after dusk. The results indicate that irradiance during measurement should be considered when using the
Minolta SPAD-502 chlorophyll meter for the estimation of crop N-status. # 1998 Annals of Botany Company
Key words : Chlorophyll meter, nitrogen, irradiance, Oxalis acetosella L., Triticum aestiŠum L., winter wheat.
INTRODUCTION
MATERIALS AND METHODS
A chlorophyll meter (Minolta SPAD-502) has been developed to estimate the nitrogen status of crops. The
instrument measures transmission of red light at 650 nm, at
which chlorophyll absorbs light, and transmission of
infrared light at 940 nm, at which no absorption occurs. On
the basis of these two transmission values the instrument
calculates a SPAD (Soil Plant Analysis Development)
value that is quite well correlated with chlorophyll content
(Wood, Reeves and Himelrick, 1993 ; Markwell, Osterman
and Mitchell, 1995). However, chloroplasts can change their
orientation within the cell in response to incident irradiance
(Britz, 1979 ; Haupt and Scheuerlein, 1990 ; Brugnoli and
Bjo$ rkman, 1992). In low irradiance the chloroplasts are
oriented along the upper and lower cell walls, thereby
maximizing light absorption, while in high irradiance they
are oriented mainly along the vertical cell walls parallel to
incident irradiance (Brugnoli and Bjo$ rkman, 1992 ; Park,
Chow and Anderson, 1996). The effect of chloroplast
orientation on leaf light transmission varies from over
100 % in shade leaves of Oxalis oregana (Brugnoli and
Bjo$ rkman, 1992) and Tradescantia Širginiana (Park et al.,
1996) to 2 % in rice (Oryza satiŠa L.) [data from Inoue and
Shibata (1974) recalculated by Brugnoli and Bjo$ rkman
(1992)]. Since there may be high variability in light
transmission caused by irradiance induced chloroplast
movement, irradiance before measurement of SPAD values
may be expected to affect results. In the present study the
effect of irradiance on SPAD values in the shade plant O.
acetosella and in winter wheat is investigated.
Oxalis acetosella plants were collected in early August from
the forest floor of a mixed forest of deciduous trees and
Picea abies near the phytotron at A/ s, Norway. The plants
were immediately taken to the laboratory, without exposing
them to high irradiance, and placed with their roots in
beakers of water in incandescent irradiance at about 5 µmol
photons m−# s−". The irradiance treatments and SPAD value
measurements were performed on the same day. Winter
wheat (Triticum aestiŠum ‘ Rudolf ’) seeds were sown in late
August in 12 cm pots at 18 °C with natural daylength in the
daylight phytotron at A/ s (59°39«). The light in the growth
chambers was natural daylight supplemented by 100 µmol
photons m−# s−" from mercury halide lamps (Philips HPI}T
400W). Measurements were made on about 6-week-old
plants.
Four experiments were performed. Chlorophyll meter
readings (SPAD values) were repeatedly taken at the centre
of the leaves throughout the experiments. Air temperature
was maintained at 18–20 °C in all experiments. Experiment
1 : O. acetosella plants were moved from incandescent
irradiance at 5 µmol photons m−# s−" to daylight chambers.
The natural late summer sunlight (approx. 1000 µmol
photons m−# s−") with the additional 100 µmol photons m−#
s−" from mercury halide lamps was filtered through one
layer of white paper so that the irradiance of the leaves was
250 µmol photons m−# s−". After 30 min the plants were
moved back to low irradiance. Chlorophyll meter measurements were taken in the same position on each leaflet of the
same ten trifoliate leaves throughout the experiment.
Experiment 2 : O. acetosella plants were moved from
incandescent irradiance at 5 µmol photons m−# s−" to another room where the leaves were oriented perpendicularly
0305-7364}98}090389­04 $30.00}0
# 1998 Annals of Botany Company
390
Hoel and Solhaug—Effect of Irradiance on Chlorophyll Estimation
RESULTS
SPAD meter readings for O. acetosella leaves decreased by
about 15 % within 30 min as the leaves were transferred
from low to high irradiance. Transferring the leaves back to
low irradiance increased SPAD values again (Fig. 1).
Exposing the leaves to various irradiances up to 250 µmol
photons m−# s−" for 30 min showed SPAD values decreased
with irradiance (Fig. 2).
SPAD values for winter wheat plants which had been
transferred from low to high irradiance also decreased with
102
SPAD value as percent of low light value
to the light direction at different distances from a mercury
halide lamp (Philips HPI}T 400W), so that they received
from 25 to 250 µmol photons m−# s−". Chlorophyll meter
measurements were taken in the same position on each
leaflet of the same ten trifoliate leaves before and after light
exposure. Experiment 3 : Winter wheat plants were placed
under 5 µmol photons m−# s−" incandescent irradiance for
one night. In the morning leaves were cut from the plants
and placed with their base in water and moved to another
room where the leaves were fixed with tape perpendicularly
to the light direction at different distances from a mercury
halide lamp (Philips HPI}T 400W) so that they received
from 12 to 200 µmol photons m−# s−" for about 2 h. They
were then moved back to low irradiance. Fifteen leaves were
exposed to each irradiance, and each leaf was measured
twice each time. The mean of these two measurements was
statistically treated as one observation. Experiment 4 : On
20 October, chlorophyll meter readings were taken throughout the whole day for winter wheat plants in a daylight
chamber. In addition to daylight, the plants received a 10 h
period with 100 µmol photons m−# s−" from mercury halide
lamps. The irradiance was determined with a Skye SKP
216Q (400–700 nm) quantum sensor for each measurement.
100
98
96
94
92
90
88
86
84
82
0
50
100
150
200
250
Irradiance (µmol photons m–2 s–1)
F. 2. SPAD values for Oxalis acetosella leaves after 30 min high
irradiance exposure as percentages of values at 5 µmol photons m−# s−"
incandescent irradiance. Each point represents 30 measurements³s.e.
(ten trifoliate leaves with one measurement on each leaflet).
irradiance, but to a lesser extent. At 200 µmol photons m−#
s−" the decrease was about 6 %, at 25 and 50 µmol photons
m−# s−" the decrease was less than 2 %, while there was no
change at 12 µmol photons m−# s−" (Fig. 3). Measurement
of SPAD values for winter wheat throughout a normal
dark-light-dark cycle in a daylight chamber showed that
SPAD values increased by about 6 % from the dark values
at the low irradiance early in the day. Thereafter, there was
a decrease of about 8 % when the irradiance was high
during the day. SPAD values first increased and then
decreased in the evening when it became dark (Fig. 4).
31
30
DISCUSSION
5 µmol photons m–2 s–1
SPAD value
32
5 µmol photons m–2 s–1
33
250 µmol photons m–2 s–1
35
34
According to Markwell et al. (1995), the SPAD-502
chlorophyll meter calculates the values shown by the
instrument (M) by means of the following equation :
29
28
27
26
25
0
20
40
60 80 100 120 140 160 180
Time (min)
F. 1. Time course of SPAD values for Oxalis acetosella leaves
transferred from low incandescent irradiance of 5 µmol photons m−#
s−" to daylight filtered through white filter paper resulting in 250 µmol
photons m−# s−", and then back to incandescent light again. Each point
represents 30 measurements³s.e. (ten trifoliate leaves with one
measurement on each leaflet).
M ¯ log [(I« }I )}(I« }I )] ¯ log [I« I )}(I« I )]
*%! *%!
'&! '&!
*%! '&!
'&! *%!
where I and I are the currents produced by the red and
'&!
*%!
infrared light beams respectively, and I«
and I«
the
'&!
*%!
currents produced by the transmitted red and infrared light.
If the leaf was an ideal optical system with only chlorophyll
absorbing light, the above equation would simplify to Mideal
¯ log (I }I« ). For example, if the transmittance of red
'&! '&!
light of a shade leaf increases by 100 % from 5 to 10 % due
to chloroplast movement, and if the transmittance of
infrared light remains unchanged, there will be a decrease in
the meter value of about 30 %. However, according to this
ideal equation, there is a fixed decrease in meter values due
to a fixed percentage increase in transmittance. The
percentage decrease in meter values will decrease with
increasing meter values.
391
Hoel and Solhaug—Effect of Irradiance on Chlorophyll Estimation
102
101
SPAD value as percent of start
100
99
98
97
96
95
200 µmol photons m–2 s–1
94
100 µmol photons m–2 s–1
93
25 µmol photons m–2 s–1
50 µmol photons m–2 s–1
12 µmol photons m–2 s–1
92
2
1
0
3
4
Time (h)
F. 3. Time course of SPAD values for Triticum aestiŠum leaves transferred from 5 µmol photons m−# s−" incandescent irradiance (time 0, value
set to 100 %) to various higher irradiances and then (indicated by arrows) back to low incandescent irradiance. Each point is the mean of 15
leaves³s.e.
45
600
SPAD value
400
43
300
42
200
41
40
PPF (µmol m–2 s–1)
500
44
100
0
0
2
4
6
8
10
12
Time (h)
F. 4. Diurnal variation in SPAD values (—E—) for Triticum
aestiŠum leaves and photosynthetic photon flux (PPF) ([[[D[[[) in
daylight growth chambers from before dawn to after dusk in October.
Each point represents 30 measurements³s.e.
The decrease in SPAD-values with irradiance was larger
and more rapid at low irradiance in the shade adapted plant
O. acetosella than in winter wheat, where irradiances up to
about 50 µmol photons m−# s−" had a relatively small effect
(Figs 2 and 3). Similarly a larger decrease in transmittance
through leaves of the shade plant O. oregana than through
leaves of the sun plant Helianthus annuus was also found
with increasing irradiance (Brugnoli and Bjo$ rkman, 1992).
In both O. acetosella and winter wheat the SPAD-values
increased again when the plants were transferred back to
low irradiance (Figs 1 and 3).
In dark adapted leaves of Helianthus annuus L. (Brugnoli
and Bjo$ rkman, 1992) and Tradescantia albiflora (Terashima
and Hikosaki, 1995) leaf light transmittance first decreased
at low irradiance. This decrease is explained by chloroplast
orientation along the upper and lower cell walls perpendicular to incident irradiance. However, with increasing
irradiance leaf light transmittance increased in both H.
annuus and T. albiflora, an effect explained by chloroplast
orientation along vertical cell walls parallel to incident
irradiation. The increase in SPAD-values for winter wheat
leaves at dawn and dusk, and the decrease in SPAD-values
at high irradiance during the day (Fig. 4) may similarly be
explained by chloroplast movement and orientation.
The effect of irradiance on SPAD-values should be
considered when SPAD-values are used for the estimation
of the nitrogen status of crops. Most crops are sun adapted
plants in which leaf transmittance is less affected by
irradiance than in shade plants (Inoue and Shibata, 1974 ;
Park et al., 1996). For example, in rice the leaf transmittance
was hardly affected by irradiance (Inoue and Shibata, 1974),
and SPAD values are therefore probably not affected by
392
Hoel and Solhaug—Effect of Irradiance on Chlorophyll Estimation
irradiance in rice. However, in winter wheat grown in
growth chambers, the SPAD-values are significantly affected
by irradiance (Figs 3 and 4). If a similar diurnal variation
occurs in the field, then time of day may affect N-status
estimates made using SPAD-values. Weather conditions
may also be important, since the SPAD values decrease with
increasing irradiance, at least below 100 µmol photons m#
s−" (Fig. 3) ; irradiance can easily drop below 100 µmol
photons m# s−" on cloudy and rainy days.
In conclusion, irradiance has been shown to affect the
chlorophyll meter readings by the Minolta SPAD-502
chlorophyll meter, thus irradiance should be considered if
the chlorophyll meter readings are to be used to estimate
crop N-status.
A C K N O W L E D G E M E N TS
The authors would like to thank Professor Ola M. Heide at
The Agricultural University of Norway, Department of
Biology and Nature Conservation and Dr Hugh Riley at
The Norwegian Crop Research Institute, division Kise for
help in correcting the manuscript.
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