Non-destructive analysis of photosynthetic pigments in cotton plants

Abstract

Analytical techniques used to extract chlorophyll from plant leaves are destructive and based on the use of organic solvents. This study proposes a non-destructive quantification of the photosynthetic pigment concentration in cotton leaves using two portable chlorophyll meters, the SPAD-502 and the CLOROFILOG 1030. After obtaining 200 leaf discs, each with an area of 113 mm(2), the greening rate in each disc was determined by the average of five readings from both meters. Immediately after measurement, 5 mL of dimethyl sulfoxide (DMSO) was added, and the samples were kept in a water bath at 70 degrees C for 30 min. After cooling, 3 mL of the liquid extract was used for analyses by spectrophotometry at 470, 646 and 663 nm. Mathematical models were adjusted from analytical results using the reading index obtained from both devices to predict the contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids. Based on these results, it was concluded that both portable chlorophyll meters are an effective way to estimate the concentration of photosynthetic pigments in cotton leaves, thus saving time, space and the resources that are often required for these analyses.

Full text

DOI: 10.4025/actasciagron.v33i4.10926
Acta Scientiarum. Agronomy Maringá, v. 33, n. 4, p. 671-678, 2011
Non-destructive analysis of photosynthetic pigments in cotton plants
Giovani Greigh Brito*, Valdinei Sofiatti, Ziany Neiva Brandão, Vivianny Belo Silva,
Franklin Magnum Silva and Dalva Almeida Silva
Centro Nacional de Pesquisa do Algodão, Empresa Brasileira de Pesquisa Agropecuária, Rod. GO-462, km 12, Cx. Postal 179,
75375-000, Santo Antonio de Goiás, Goiás, Brazil. *Author for correspondence. E-mail: giovani@cnpa.embrapa.br
ABSTRACT. Analytical techniques used to extract chlorophyll from plant leaves are
destructive and based on the use of organic solvents. This study proposes a non-destructive
quantification of the photosynthetic pigment concentration in cotton leaves using two
portable chlorophyll meters, the SPAD-502 and the CLOROFILOG 1030. After obtaining
200 leaf discs, each with an area of 113 mm2, the greening rate in each disc was determined
by the average of five readings from both meters. Immediately after measurement, 5 mL of
dimethyl sulfoxide (DMSO) was added, and the samples were kept in a water bath at 70ºC
for 30 min. After cooling, 3 mL of the liquid extract was used for analyses by
spectrophotometry at 470, 646 and 663 nm. Mathematical models were adjusted from
analytical results using the reading index obtained from both devices to predict the contents
of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids. Based on these results, it
was concluded that both portable chlorophyll meters are an effective way to estimate the
concentration of photosynthetic pigments in cotton leaves, thus saving time, space and the
resources that are often required for these analyses.
Keywords: Gossypium hirsutum, chlorophyll, carotenoids, cotton.
RESUMO. Análise não destrutiva dos pigmentos fotossintéticos em plantas de
algodoeiro. Técnicas analíticas empregadas na extração de clorofila em plantas são destrutivas
e fundamentam-se no uso de solventes orgânicos. Este estudo propõe a quantificação não
destrutiva da concentração de pigmentos fotossintéticos em folhas de algodoeiro utilizando os
medidores portáteis de clorofila SPAD-502 e CLOROFILOG 1030. Com as folhas coletadas
foram elaborados 200 discos foliares com área de 113 mm2. A determinação do índice de
esverdeamento em cada disco foi realizada por meio da média de cinco leituras com ambos
clorofilômetros portáteis e imediatamente após a determinação, adicionaram-se 5 mL de
Dimetil sulfóxido (DMSO). Os discos foram mantidos em banho-maria a temperatura de
70ºC por um período de 30 min. Após o resfriamento do extrato líquido, uma alíquota de
3,0 mL foi utilizada para leitura utilizando espectrofotometria a 470, 646 e 663 nm. A partir
dos resultados analíticos obtidos foram ajustados modelos matemáticos utilizando-se o índice
das leituras efetuadas por ambos os equipamentos para estimar os teores de clorofila a, clorofila
b, clorofila total e carotenóides. Considerando os resultados obtidos conclui-se que ambos
medidores portáteis de clorofila poderão ser utilizados para estimar a concentração dos
pigmentos fotossintéticos em folhas de algodoeiro, economizando tempo, espaço e recursos
comumente demandados nessas análises.
Palavras-chave: Gossypium hirsutum, clorofila, carotenóides, algodão.
Introduction
There are important factors related to
photosynthetic efficiency in plants, such as the
concentration and composition of chloroplast
pigments that affect plant growth and their
adaptability to environments with different
luminosities (DAI et al., 2009).
The production of dry matter by crop species
and their ability for abiotic stress tolerance has been
influenced by the amount of chlorophyll (Chl)
present, due to the vital relationship of this pigment
with the photosynthetic process (DAWSON et al.,
2003; LIETH; WHITTAKER, 1975). Losses in
chlorophyll content are associated with damaging
environmental factors, such that variations in the
total chlorophyll/carotenoids ratio are good
indicators of plant injury (HENDRY; PRICE, 1993;
KARA; MUJDECI, 2010).
The determination of the leaf chlorophyll
content is a common procedure for plant
scientists. Destructive techniques have been
traditionally used for the determination of
chlorophyll content in stands of vegetation. In

672 Brito et al.
Acta Scientiarum. Agronomy Maringá, v. 33, n. 4, p. 671-678, 2011
general, these techniques involve very laborious
and destructive sampling plus various analytical
protocols (LIETH; WHITTAKER, 1975;
TUCKER, 1977). These methods use organic
solvents that include acetone (BRUISNA, 1961;
MAcKINNEY, 1941), dimethyl sulfoxide
(DMSO) (HISCOX; ISRAELSTAM, 1979),
methanol, N, N-dimethyl formamide and
petroleum ether (INSKEEP; BLOOM, 1985;
LICHTENTHALER; WELLBURN, 1983).
During the extraction and dilution processes,
significant pigment loss can take place, leading to
a high variability in the results. Shoaf and Lium
(1976) modified the extraction methodology
using DMSO, thus eliminating the squashing and
centrifuging stages. This method allowed for the
extension of the storage period for the extracted
pigment, so that spectrophotometric analyses need
not be performed immediately after extraction.
Chlorophyll meters are extensively used in
agriculture; they quickly estimate the chlorophyll
content of leaves with a hand-held device that
measures the leaf absorbance in two different
wavelength regions using two light emitting diodes
(LEDs). The chlorophyll meter Soil Plant Analysis
Development (SPAD-502) is a simple and portable
diagnostic tool that measures the greenness or the
relative chlorophyll concentration of leaves
(KARIYA et al., 1982; TORRES-NETTO et al.,
2005). It provides instantaneous and non-destructive
readings on plants based on the quantification of the
intensity of absorbed light by the tissue sample using
a red LED (wavelength peak is ~650 nm) as a
source. An infrared LED, with a central wavelength
emission of approximately 940 nm, acts
simultaneously with the red LED to compensate for
the leaf thickness (MINOLTA CAMERA Co. Ltd.,
1989).
Another device used to estimate chlorophyll
concentration is the Clorofilog 1030 chlorophyll
meter, which has three LEDs working at 635, 660
and 850 nm, with the last wavelength used for the
normalization of readings. This device might
provide a substantial savings in time, space and
resources. To determine the amount of chlorophyll
in a sample, the mathematical relationship between
the meter readings and the chlorophyll
concentration in the tissue sample must be made.
However, to determine the chlorophyll
concentration of a sample with a chlorophyll meter,
the mathematical relationship between meter
readings and the chlorophyll concentration in the
tissue sample must be ascertained. The chlorophyll
concentration, or leaf greenness, is affected by many
factors. One such factor is the status of nitrogen (N)
in the leaves (KARA; MUJDECI, 2010). A positive
correlation between leaf N or the N fertilization rate
and chlorophyll content has been well documented
for a large number of plant species, and it has been
investigated for a rapid determination of the N status
using Chl meters in most major crops, including corn
(Zea mays L.), rice (Oryza sativa L.), wheat (Triticum
aestivum L.) and cotton (Gossypium hirsutum L.), as well
as numerous other plant species (BULLOCK;
ANDERSON, 1998; CHANG; ROBISON, 2003;
EVANS, 1989; LIN et al., 2010; MAUROMICALE
et al., 2006; NAGESWARA RAO et al., 2001;
NTAMATUNGIRO et al., 1999; PENG et al., 1993;
REEVES et al., 1993; WU et al., 1998).
Carotenoids are integral constituents of the
thylakoid membrane and are usually well associated
with many of the proteins that constitute the
photosynthetic apparatus (SIKUKU et al., 2010).
They represent an important role in the light-
harvesting complex, as well as in the
photoprotection of the photosystems (PSs). Some
reports show that these compounds are very
important in the preservation of the photosynthetic
apparatus against photodamage by their
interconversion with xanthophyll molecules (ORT,
2001; YOUNG et al., 1997). In the xanthophyll
cycle, violaxanthin undergoes a de-epoxidation to
give rise to anteroxanthin and, finally, zeaxanthin
(HAVAUX, 1988). Zeaxanthin participates in the
regulation of the heat dissipation of PSII energy
when this photosystem has an energetic overload
(ORT, 2001). Therefore, an indirect and non-
destructive quantification of the total content of
carotenoids is of great importance for many related
studies.
The main objectives of this study were the
following: to assess the cotton chlorophyll
composition and establish a possible correlation
between the photosynthetic pigments extracted in
DMSO with readings obtained by both chlorophyll
meters; and to verify the relationship between these
characteristics in the leaf tissue of Upland Cotton
cultivated under field conditions.
Material and methods
Plant material and growth conditions
Cotton plant leaves (BRS 187 8H) were collected
when the crop was at the height of the flowering
period, characterized as the F4 cotton development
stage (MARUR; RUANO, 2001; ROSOLEM,
2007). The experiment was carried out at the
experimental station of the National Center of
Cotton Research, located in Apodi, Rio Grande do

Evaluation of chlorophyll meters for cotton management 673
Acta Scientiarum. Agronomy Maringá, v. 33, n. 4, p. 671-678, 2011
Norte State, Brazil (5º37'22" S; 37º48'58" W; 131 m
of altitude). The region climate is characterized as
warm tropical and semi-arid, with a predomination
of BSw´h´ type (KÖPPEN; GEIGER, 1928). The
soil is classified as Eutrophic Cambisol. The
planting date was September 23, 2008. The
chlorophyll extraction and greenness reading
indexes were obtained from leaves harvested at the
base of the petiole and placed in plastic zip-loc bags
that were kept in the dark and cool until arrival at
the laboratory. All samples were processed within
approximately 2 hours after being gathered in the
field.
Chlorophyll meter readings
Leaf disks were randomly sampled from leaves
using a borer with a diameter of 12.0 mm. Five
readings obtained by both portable chlorophyll
meters (SPAD-502 by Minolta, Japan and
CLOROFILOG 1030 by Falker, Brazil) on each disc
from individual leaves were averaged.
Approximately 200 leaf discs were used, and the
values obtained by the meters varied from 4 to 60,
making the maximum amplitudes between value
readings.
Photosynthetic pigment analysis
After obtaining the meter readings, the
chlorophyll was extracted from the leaf disks using
the Hiscox and Israelstam (1979) procedure. Each
disc was cut into smaller pieces and placed in a test
tube containing 5 mL of dimethyl sulfoxide
(DMSO). All samples were incubated at 70ºC for
30 min. (HISCOX; ISRAELSTAM, 1979) until all
of the visible green pigmentation disappeared. After
cooling, a 3-mL aliquot of the chlorophyll extract
was transferred to a cuvette for the determination of
the chlorophyll absorbance using a
spectrophotometer at 470, 646 and 663 nm.
Absorption measurements were used to quantify the
chlorophyll a, chlorophyll b, and total chlorophyll
concentrations, based on the equations reported by
Wellburn (1994).
Data analyses
Analysis of variance (ANOVA) (p < 0.05) was
applied to the data, and linear regression analyses
were made. The mathematical equations were
adjusted with a high coefficient of determination.
The readings for the greenness indexes were used as
the dependent variable, while the pigment
concentrations extracted by the classical method
were used as the independent variable. Data analyses
were conducted using SigmaPlot 10.0 software in
order to fit the suitable mathematical equations to all
of the analyzed variables. A Pearson correlation
analysis between the two portable chlorophyll
meters readings was conducted.
Results and discussion
Despite the fact that the two portable meters
provided different values for the chlorophyll
measurements, we observed a high correlation
between their data (Figure 1). The Clorofilog 1030
(Falker Agricultural Automation) chlorophyll meter
showed higher values than the SPAD-502
(MINOLTA CAMERA Co. Ltd., 1989) meter. It
was observed that this difference was higher in
leaves that presented less chlorophyll content
spectrophotometrically, namely, with the lower
readings with the devices. Consequently, it was
necessary to perform distinct adjustments in the
mathematical models for the prediction of
chlorophyll and carotenoid content by each portable
meter.
We suggest that it is likely that such differences
occurred because these two devices operate in
different wavelength ranges. According to Markwell
et al. (1995), the chlorophyll meter developed by
Minolta uses two LEDs in the bands of 650 and 940
nm and a photodiode detector to measure
sequentially the transmittance of red and infrared
light through the leaves. In contrast, the Clorofilog
1030 functions with LEDs in wavelengths of 635,
660 and 880 nm.
SPAD reading (Minolta)
0 10203040506070
Clorofilog reading (Falker)
0
10
20
30
40
50
60
70
99.0
94.068.5
ˆ
=
+=
r
XY
Figure 1. The relationship between SPAD-502 readings
(MINOLTA CAMERA Co. Ltd., 1989) and Clorofilog 1030
readings (Falker Automation) in Gossypim hirsutum leaves.
Figures 2 and 3 show the relationships between
the readings obtained in cotton leaves by the two
chlorophyll meters and the concentrations of
chlorophylls a and b, respectively. The relationship
between the chlorophyll readings from both
portable meters and the contents of chlorophyll a

674 Brito et al.
Acta Scientiarum. Agronomy Maringá, v. 33, n. 4, p. 671-678, 2011
and chlorophyll b was more readily expressed with a
quadratic model. Determination coefficients of the
adjusted models were 0.90 and 0.91 for chlorophyll
a and 0.82 and 0.80 for chlorophyll b using the
SPAD 502 and the Clorofilog 1030 meters,
respectively.
The relationship between the chlorophyll
readings in both portable meters and the
concentrations of carotenoids were fit in a quadratic
model, and an R2 value of 0.79 was obtained for
both meters (Figures 4a and 5a). The relationship
between the readings and total chlorophyll are
presented in Figures 4b and 5b, and a high R2 value
was obtained. The coefficient of determination for
the adjusted models of total chlorophyll content was
0.91 for both of the chlorophyll meters.
The relationship between the photosynthetic
pigment concentration and the chlorophyll readings
have been established for several species of plants, such
as the total chlorophyll in Glycine max and Zea mays
(MARKWELL et al., 1995) and Chl a, b, total Chl and
carotenoids in Carica papaya L. (TORRES NETO
et al., 2002) and Coffea canephora Pierre (TORRES
NETO et al., 2005). The relationship between the
chlorophyll meter readings obtained by the devices and
the concentrations of photosynthetic pigments was
adequately represented for cotton by the quadratic
mathematical model, suggesting that this species has a
similar relationship as the leaves of wheat, rice, soybean
(MONJE; BUGBEE, 1992) and coffee (TORRES
NETO et al., 2005). In some species, the linear and
exponential models have also been adjusted to express
these relationships (TORRES NETO et al., 2002).
Figures 6a and 7a show the relationship between
total chlorophyll/carotenoids and the chlorophyll meter
readings obtained by the SPAD 502 and Clorofilog
1030 devices, respectively. The readings obtained by
both chlorophyll meters allowed for the estimation of
these relationships between chlorophyll and
carotenoids via an indirect, though highly precise
method. The quadratic mathematical model provided a
better representation, with determination coefficients
of 0.91 and 0.92 for the SPAD-502 and Clorofilog
1030 meters, respectively.
SPAD reading
0 10203040506070
Chl a (μ mol. m-2)
0
100
200
300
400
500
600
90.0=
09.0+18.3+18.10-=
ˆ
2
2
R
XXY
(a)
SPAD reading
0 10203040506070
Chl b (μ mol. m-2)
0
25
50
75
100
125
150
82.0=
02.0+22.0+15.24=
ˆ
2
2
R
XXY
(b)
Figure 2. The relationships between SPAD-502 readings, chlorophyll a (Chl a) (a) and chlorophyll b (Chl b) (b) in Gossypim hirsutum leaves.
Clorofilog readin g
0 10203040506070
Chl a (μ mol. m-2)
0
100
200
300
400
500
600
91.0=
07.0+62.4+23.56-=
ˆ
2
2
R
XXY
(a)
Clorofilog readin g
0 10203040506070
Chl b (μ mol. m-2)
0
25
50
75
100
125
150
80.0=
018.0+31.0+27.20=
ˆ
2
2
R
XXY
(b)
Figure 3. The relationships between Clorofilog 1030 readings, chlorophyll a (c) and chlorophyll b (Chl b) in Gossypim hirsutum leaves.

Evaluation of chlorophyll meters for cotton management 675
Acta Scientiarum. Agronomy Maringá, v. 33, n. 4, p. 671-678, 2011
SPAD reading
0 10203040506070
Car (μ mol. m-2)
0
50
100
150
200
250
300
350
400
79.0=
05.0+16.0+15.95=
ˆ
2
2
R
XXY
(a) SPAD reading
0 10203040506070
Total chl (μ mol. m-2)
0
100
200
300
400
500
600
700
91.0=
11.0+41.3+97.13=
ˆ
2
2
R
XXY
(b)
Figure 4. The relationships between SPAD-502 readings, carotenoid (Car) concentration (a) and total chlorophyll (total Chl) (b) in
Gossypim hirsutum leaves.
Clorofilog reading
0 10203040506070
Car (μ mol. m-2)
0
50
100
150
200
250
300
350
400
79.0=
05.0+06.0+67.90=
ˆ
2
2
R
XXY
(a)
Clorofilog readin g
0 10203040506070
Total chl (μ mol. m-2)
0
100
200
300
400
500
600
700
91.0=
09.0+94.4+95.35-=
ˆ
2
2
R
XXY
(b)
Figure 5. The relationships between Clorofilog 1030 readings, carotenoid (Car) concentration (a) and total chlorophyll (total Chl) (b) in
Gossypim hirsutum leaves.
SPAD reading
0 10203040506070
Total chl / car
0.0
0.5
1.0
1.5
2.0
2.5
91.0=
0004.0-05.0+15.0=
ˆ
2
2
R
XXY
(a) SPAD reading
0 10203040506070
Chl a / b
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
73.0=
0011.0-15.0+09.0-=
ˆ
2
2
R
XXY
(b)
Figure 6. The relationships between SPAD- 502 readings, the total Chl/Car ratio (a) and the Chl a/b ratio (b) in Gossypim hirsutum leaves.
Plants with SPAD readings lower than 40
presented reduction of the relationship total
chlorophyll/carotenoids. The same effect was also
observed when a SPAD 502, with values lower than
40, was used for leaves of Carica papaya L. (TORRES
NETO et al., 2002) and Coffea canephora Pierre
(TORRES NETO et al., 2005). This may be due to
the onset of leaf senescence (BUCKLAND et al.,

676 Brito et al.
Acta Scientiarum. Agronomy Maringá, v. 33, n. 4, p. 671-678, 2011
1991). The relationship between chlorophyll and
carotenoids has been much less used, although this
ratio may be considered a good indicator to
distinguish between natural senescence and
senescence as a result of environmental injuries,
such as desiccation in mosses (BUCKLAND et al.,
1991) and the occurrence of water deficit in plants in
bloom (SEEL et al., 1992; SIKUKU et al., 2010).
Measurements of less than 40 indicate the
beginning of a possible reduction in photosynthetic
processes. This effect was also observed by Torres
Neto et al. (2005) in coffee plants. Additionally,
this relationship has been considered a good
indicator of disturbances in plants that have been
caused by environmental factors (HENDRY;
PRICE, 1993).
Figures 6b and 7b show the relationship
between chlorophyll a/b and the chlorophyll meter
readings from the SPAD 502 and Clorofilog 1030
meters, respectively. Similar to the other
characteristics analyzed, the quadratic mathematical
model best fit the data, exhibiting determination
coefficients above 0.73 and 0.76 for the SPAD and
Clorofilog meters, respectively. Analogous with the
behavior of the total chlorophyll/carotenoids ratio,
a dramatic reduction in values was observed when
readings were below 40 for the chlorophyll a/b
ratio.
Chlorophyll a is more strongly degraded than
chlorophyll b (WOLF, 1956), which may explain
the reduction of the chlorophyll a/b ratio when the
chlorophyll meter readings were below 40 (Figure
2b). These low readings may occur in shaded
leaves; because the total chlorophyll content per
unit of leaf area is lower in leaves that are exposed
to high irradiance, while the ratio between
chlorophyll a/b is larger, when compared with
leaves grown under shaded conditions. The effect
of the photon flux density on the chlorophyll a/b
ratio is one of the most striking features between
plants growing under sunny or shady conditions
(ANDERSON, 1986; BOARDMAN, 1977).
Because of this response to different light
intensities, the chlorophyll a/b ratio has been
proposed as a bioassay to analyze the irradiance
level to which a plant was subjected (DALE;
CAUSTON, 1992). In fact, the chlorophyll
content and the chlorophyll a/b ratio are responsive
to changes within the mesophyll of individual
leaves (CUI et al., 1991; TERASHIMA et al.,
1986).
The chloroplasts of leaves grown in the shade
develop a higher proportion of thylakoids
compared to the volume of the stroma, with both
larger thylakoids and more thylakoids per granum
(ANDERSON, 1986; BOARDMAN, 1977). While
the relative proportion of chlorophyll associated
with the compound complex of Photosystem I and
the reaction center of Photosystem II decreases
with a reduction in the a/b ratio, the relative
proportion of chlorophyll associated with the a/b
light-collecting protein complex increases
(LEONG; ANDERSON, 1984). The complex
light collector (LHC2) has a lower chlorophyll a/b
ratio than the other proteins linked to the
chlorophyll molecules associated with Photosystem
II, because LHC2 contains most of the chlorophyll
b (GREEN; DURNFORD, 1996). Therefore, the
chlorophyll a/b ratio may be useful as an indicator
of the chloroplast composition. Some authors have
used this ratio as an indicator of leaf N
partitioning, based on the positive relationship
between chlorophyll a/b and the rate of light
collected by the chlorophyll-protein complex of
Photosystem II (KITAJIMA; HOGAN, 2003;
TERASHIMA et al. 1986).
Clorofilog readin g
0 10203040506070
Total chl / car
0,0
0,5
1,0
1,5
2,0
2,5
92.0=
0006.0-08.0+41.0=
ˆ
2
2
R
XXY -
(a)
Clorofilog reading
0 10203040506070
Chl a / b
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
76.0=
0017.0-21.0+61.1=
ˆ
2
2
R
XXY -
(b)
Figure 7. The relationships between Clorofilog 1030 readings, the total Chl/Car ratios (a) and Chl a/b ratios (b) in Gossypim hirsutum
leaves.

Evaluation of chlorophyll meters for cotton management 677
Acta Scientiarum. Agronomy Maringá, v. 33, n. 4, p. 671-678, 2011
Conclusion
In general, it was observed that the use of the
portable chlorophyll meters, SPAD-502 and 1030
Clorofilog, produced results associated with
empirical models and allowed for a quick prediction
of the concentration of photosynthetic pigments in
the leaves of cotton, with high accuracy and without
the use of chemical reagents and extensive
laboratory protocols.
References
ANDERSON, J. M. Photoregulation of the composition,
function, and structure of thylakoid membranes. Annual
Review of Plant Physiology, v. 37, p. 93-136, 1986.
BOARDMAN, N. K. Comparative photosynthesis of sun
and shade plants. Annual Review of Plant Physiology,
v. 28, p. 355-377, 1977.
BRUISNA, J. A comment on the spectrophotometric
determination of chlorophyll. Biochimica et
Biophysica Acta, v. 52, p. 576-578, 1961.
BUCKLAND, S. M.; PRICE, A. H.; HENDRY, G. A. F.
The role of ascorbate in drought-treated Cochlearia atlantica
Pobed. and Armeria maritime (Mill.) Willd. New
Phytologist, v. 119, n. 1, p. 155-160, 1991.
BULLOK, D. G.; ANDERSON, D. S. Evaluation of the
minolta SPAD – 502 chlorophyll meter for nitrogen
management in corn. Journal of Plant Nutrition, v. 21,
n. 3, 741-755, 1998.
CHANG, S. X.; ROBISON, D. S. Nondestructive and
rapid estimation of hardwood foliar nitrogen status using
the SPAD-502 chlorophyll meter. Forest Ecology and
Management, v. 181, n. 3, p. 331-338, 2003.
CUI, M.; VOGELMANN, T. C.; SMITH, W. K.
Chlorophyll and light gradients in sun and shade leaves of
Spinacia oleracea. Plant, Cell and Environment, v. 14,
n. 5, p. 493-500, 1991.
DAI, Y.; SHEN, Z.; LIIU, Y.; WANG, L.; HANNAWAY,
K.; LU, H. Effects of shade treatments on the
photosynthetic capacity, chlorophyll fluorescence, and
chlorophyll content of Tetrastigma hemsleyanum Diels et
Gilg. Environmental and Experimental Botany, v. 65,
n. 2-3, p. 177-182, 2009.
DALE, M. P.; CAUSTON, D. R. Use of chlorophyll a/b
ratio as a bioassay for the light environment of a plant.
Functional Ecology, v. 6, n. 2, p. 190-196, 1992.
DAWSON, T. P.; NORTH, P. R. J.; PLUMMER, S. E.;
CURRAN, P. J. Forest ecosystem chlorophyll content:
implications for remotely sensed estimates of net primary
productivity. International Journal of Remote
Sensing, v. 24, n. 3, p. 611-7, 2003.
EVANS, J. R.: Photosynthesis and nitrogen relationships
in leaves of C3 plants. Oecologia, v. 78, n. 1, p. 9-19,
1989.
GREEN, B. R.; DURNFORD, D. G. The chlorophyll-
carotenoid proteins of oxygenic photosynthesis. Annual
Review of Plant Physiology and Plant Molecular
Biology, v. 47, p. 685-714, 1996.
HAVAUX, M. Carotenoids as membrane stabilizers in
chloroplasts. Trends in Plant Science, v. 3, n. 4,
p. 147-151, 1988.
HENDRY, G. A. F.; PRICE, A. H. Stress indicators:
chlorophylls and carotenoids. In: HENDRY, G. A. F.;
GRIME, J. P. (Ed.). Methods in comparative plant
ecology. London: Chapman & Hall, 1993. p. 148-152.
HISCOX, J. D.; ISRAELSTAM, G. F. A method for the
extraction of chlorophyll from leaf tissue without
maceration. Canadian Journal of Botany, v. 57, n. 12,
p. 1332-1334, 1979.
INSKEEP, W. P.; BLOOM, P. R. Extinction coefficients
of chlorophyll a and b in N.N-dimethylformamide and
80% acetone. Plant Physiolology, v. 77, n. 2, p. 483-485,
1985.
KARA, B.; MUJDECI, M. Influence of late-season
nitrogen application on chlorophyll content and leaf area
index in wheat. Scientific Research and Essays, v. 5,
n. 16, p. 2299-2303, 2010.
KARIYA, K.; MATSUZAKI, A.; MACHIDA, H.
Distribution of chlorophyll content in leaf blade of rice
plant. Japanese Journal of Crop Science, v. 51, n. 1,
p. 134-135, 1982.
KITAJIMA, K.; HOGAN, K. P. Increases of chlorophyll
a/b ratios during acclimation of tropical woody seedlings
to nitrogen limitation and high light. Plant Cell and
Environment, v. 26, n. 6, p. 857-865, 2003.
KÖPPEN, W.; GEIGER, R. Klimate der Erde. Gotha:
Verlag Justus Perthes, 1928.
LEONG, T. Y.; ANDERSON, J. M. Adaptation of the
thylakoid membranes of pea chloroplasts to light
intensities. I. Study on the distribution of chlorophyll
protein complexes. Photosynthesis Research, v. 5, n. 2,
p. 105-115, 1984.
LICHTENTHALER, H. K.; WELLBURN, A. R.
Determinations of total carotenoids and chlorophylls a
and b of leaf extracts in different solvents. Biochemical
Society Transactions, v. 11, n. 3, p. 591-592, 1983.
LIETH, H.; WHITTAKER, R. H. Primary production
of the biosphere. New York: Springer-Verlag, 1975.
LIN, F. F.; QIU, L. F.; DENG, J. S.; SHI, Y. Y.; CHEN,
L. S.; WANG, K. Investigation of SPAD meter-based
indices for estimating rice nitrogen status. Computers
and Electronics in Agriculture, v. 71, s. 1, p. S60-S65,
2010.
MAcKINNEY, G. Absorption of light by chlorophyll
solutions. The Journal of Biological Chemistry,
v. 140, p. 315-322, 1941.
MARKWELL, J.; OSTERMAN, J. C.; MITCHELL, J. L.
Calibration of the Minolta SPAD-502 leaf chlorophyll
meter. Photosynthesis Research, v. 46, n. 3, p. 467-472,
1995.
MARUR, C. J.; RUANO, O. A reference system for
determination of developmental stages of upland cotton.
Revista de Oleaginosas e Fibrosas, v. 5, n. 2,
p. 313-317, 2001.
MAUROMICALE, G.; IERNA, A.; MARCHESE, M.
Chlorophyll fluorescence and chlorophyll content in

678 Brito et al.
Acta Scientiarum. Agronomy Maringá, v. 33, n. 4, p. 671-678, 2011
field-grown potato as affected by nitrogen supply,
genotype, and plant age. Photosynthetica, v. 44, n. 1,
p. 76-82, 2006.
MINOLTA CAMERA Co. Ltd., Chlorophyll meter
SPAD-502. Instruction Manual. Osaka, Minolta:
Radiometric Instruments Divisions, 1989.
MONJE, O. A.; BUGBEE, B. Inherent limitations of
nondestructive chlorophyll meteres: A comparasion of
two types of meteres. HortScience, v. 27, n. 1, p. 69-71,
1992.
NAGESWARA RAO, R. C.; TALWAR, H. S.; WRIGHT,
G. C. Rapid assessment of specific leaf area and leaf
nitrogen in peanut (Arachis hypogaea L.) using a chlorophyll
meter. Journal of Agronomy and Crop Science,
v. 186, n. 3, p. 175-182, 2001.
NTAMATUNGIRO, S.; NORMAN, R. J.; McNEW, R.
W.; WELLS, B. R. Comparison of plant measurements for
estimating nitrogen accumulation and grain yield by
flooded rice. Agronomy Journal, v. 91, n. 4, p. 676-685,
1999.
ORT, D. When there is too much light. Plant
Physiology, v. 125, n. 1, p. 29-32, 2001.
PENG, S.; GARCÍA, F. V.; LAZA, R. C., CASSMAN, K.
G. Adjustment for specific leaf weight improves
chlorophyll meter’s estimate of rice leaf nitrogen content.
Agronomy Journal, v. 85, n. 5, p. 987-990, 1993.
REEVES, D. W.; MASK, P. L.; WOOD, C. W.;
DELANEY, D. P. Determination of wheat nitrogen status
with a hand-held chlorophyll meter: influence of
management practices. Journal of Plant Nutrition,
v. 16, n. 4, p. 781-796, 1993.
ROSOLEM, C. A. Fenologia e ecofisiologia no manejo do
algodoeiro. In: FREIRE, E. C. (Ed.). Algodão no
Cerrado do Brasil. Brasília: Abrapa, 2007. p. 649-688.
SEEL, W. E.; HENDRY, G. A. F.; LEE, J. A. The
combined effect of desiccation and irradiance on mosses
form xeric and hydric habitats. Journal of Experimental
Botany, v. 43, n. 8, p. 1023-1030, 1992.
SHOAF, T. W.; LIUM, B. W. Improved extraction of
chlorophyll a and b from algae using dimethyl sulphoxide.
Limnology and Oceanography, v. 21, n. 6, p. 926-928,
1976.
SIKUKU, P. A.; NETONDO, G. W.; ONYANGO, J.
C.; MUSYIMI, D. M. Chlorophyll fluorescence, protein
and chlorophyll content of three nerica rainfed rice
varieties under varying irrigation regimes. ARPN Journal
of Agricultural and Biological Science, v. 5, n. 2,
p. 19-25, 2010.
TERASHIMA, I.; SAKAGUCHI, S.; HARA, N. Intra-leaf
and intracellular gradients in chloroplast ultrastructure of
dorsiventral leaves illuminated from the adaxial or abaxial
side during their development. Plant and Cell
Physiology, v. 27, n. 6, p. 1023-1031, 1986.
TORRES NETTO, A.; CAMPOSTRINI, E.;
OLIVEIRA, J. G.; SMITH, R. E. B. Photosynthetic
pigments, nitrogen, chlorophyll a fluorescence and SPAD-
502 readings in coffee leaves. Scientia Horticulturae,
v. 104, n. 2, p. 199-209, 2005.
TORRES NETTO, A.; CAMPOSTRINI, E.;
OLIVEIRA, J. G.; YAMANISHI, O. K. Portable
chlorophyll meter for the quantification of photosynthetic
pigments, nitrogen and the possible use for assessment of
the photochemical process in Carica papaya. Brazilian
Journal of Plant Physiolology, v. 14, n. 3, p. 203-210,
2002.
TUCKER, C. J. Asymptotic nature of grass canopy
spectral reflectance. Applied Optics, v. 16, n. 5,
p. 1151-1156, 1977.
WELLBURN, A. R. The spectral determination of
chlorophylls a and b, as well as total Carotenoids, using
various solvents with spectrophotometers of different
resolution. Journal of Plant Physiology, v. 144, n. 3,
p. 307-313, 1994.
WOLF, F. T. Changes in chlorophylls a and b in autumn
leaves. American Journal of Botany, v. 43, n. 9, p. 714-718,
1956.
WU, F.; WU L.; XU, F. Chlorophyll meter to predict
nitrogen sidedress requirements for short-season cotton
(Gossypium hirsutum L.). Field Crops Research, v. 56,
p. 309-314, 1998.
YOUNG, A.; PHILLIP, D.; SAVILL, J. Carotenoids in
higher plant photosynthesis. In: PESSARAKLI, M. (Ed.).
Handbook of photosynthesis. New York: Marcel
Dekker, 1997. p. 575-596.
Received on August 18, 2010.
Accepted on November 5, 2010.
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