CANADAN JOURML OF PI.
AI\IT SCIENCE
Vol. 57 January 1977 No. I
THE RELATIONSHIP BETWEEN CHLOROPHYLL CONTENT AND RATE
OF PHOTOSYNTHESIS IN SOYBEANS
B. R. BUTTERY and R. L BUZZELL
Research Station, Agricuhure Canada, Harrow, Ontario NOR IG0 .
Received 10 May 1976, accepted 29 July 1976'
BurrBnv, B. R. aNo Buzzsrr, R. I. 1977. The relationship between
Can. J. Plant Sci. Downloaded from cdnsciencepub.com by 49.34.71.181 on 12/26/23
chlorophyll content and rate of photosynthesis in soybeans. Can. J. Plant Sci.
57: l-5.
Photosynthetic rate of soybeans (on a leaf area basis, Pi estimated from the
incorporation of 1aCO2 under field conditions was highly correlated with chlorophyll
content of the side leaflets of the same leaves. Among a collection of 48 cultivars,
the linear regression of P6 on chlorophyll content accounted for 44Va of the
variation, whereas with a selection of genotypes with various mutant chlorophyll
genes, the regression accounted for 8lVa. When the data tbr the two tests were
re-calculated relative to the check cv. Altona, a quadratic equation between Pa and
chlorophyll accounted for nearly 9O4o of the variation. When photosynthetic rate
was expressed on a unit chlorophyll basis (P"), no significant differences among
lines were established in the cultivar test. In the mutants test, significant differences
in P" were established with higher values of P" associated with lower chlorophyll
contents; a linear regression accounted for 457o of the variation. Transformation of
the data from both experiments relative to Altona allowed the fitting of a common
regression line (quadratic) which accounted for 637o of the variation. We suggest
that initial screening of progenies in a breeding program for high photosynthetic rate
could be done by measuring chlorophyll content'
Le taux photosynth6tique du soja (surface foliaire P6), estim6 ir partir de
I'incorporation de 1aCO2 au champ a montr6 une forte correlation avec la teneur en
chlorophylle des folioles lat6rales des mdmes feuilles' Dans un groupe de 48
cultivars, la r6gression lin6aire de Pa sur la teneur en chlorophylle arepresente 44qo
de la variation, alors qu'elle en repr6sentait 817o dans une s6lection de g6notypes
poss6dant divers gdnes mutants de la chlorophylle. Aprbs ce calcul des donn6es des
deux essais en regard du cultivar t6moin Altona, 1'6quation quadratique entre Pa et la
teneur en chlorophytlb a rendu compte d'environ 907o de la variation. Dans I'essai
des cultivars, on n'a constat6 aucune diff6rence significative entre les lign6es
lorsque le taux photosynth6tique 6tait exprim6 par unit6 de chlorophylle (P"). Dans
l'essai des mutants, on a observ6 des diff6rences significatives du P", les valeurs plus
6lev6es 6tant associ6es aux teneurs plus faibles en chlorophylle; la r6gression
lin6aire a repr6sent6 457o de la variation. La transformation des donn6es provenant
des deux exp6riences en regard du cultivar Altona a permis d'ajuster une ligne de
r6gression commune (quadratique) qui a compt6 pour 637o de la variation. Tout
porte d croire qu'un premier tri de g6n6rations, dans le cadre d'un programme de
s6lection pour le taux photosynth6tique, pourrait se faire par la d6termination de la
teneur en chlorophylle.
The relationship between chlorophyll con- been the subject of some conflicting opin-
tent of leaves and photosynthetic rate has ions. Haberlandt (1914) showed a close
Can. J. Plant Sci.57: l-5 (Jan. 1977) relationship between photosynthetic activity
I
CANADIAN JOURNAL OF PLANT SCIENCE
and numbers of chloroplasts per unit of leaf (incident quanta) for light to be saturating; it
area. However, Willsthtter and Stoll (1918) is only at low light levels that the extra light
showed that yellow cultivars of Ulmus and absorbed by a high chlorophyll content
Sambucus could assimilate at rates similar becomes important. The results of Sesftik
to those of green varieties of the same (1966) do not support this hypothesis; his
species. Benedict (1972) calculated that results indicate that the correlation between
low-chlorophyll mutants of several flower- rate of photosynthesis and chlorophyll
ing plants (cotton, pea, peanut, soybean, content was higher at high irradiance than at
and tobacco) have photosynthetic rates per low.
milligram of chlorophyll that were 2-ll Many crops are never light-saturated;
Can. J. Plant Sci. Downloaded from cdnsciencepub.com by 49.34.71.181 on 12/26/23
times higher than that of the normal green even individual leaves of many plants are
types. not fully saturated in full sunlight (e.g.
Hesketh (1963) showed that species can Hesketh (1963) for maize; Bowes et al.
vary greatly in rate of photosynthesis and (1972) for soybeans). Thus one could
that this variation is not related to suppose that a higher level of chlorophyll
chlorophyll content. Maize leaves may content could further increase light absorp-
contain 2.1-3.0 mg chlorophyll/dmz and tion and, therefore, photosynthetic rate. We
have photosynthetic rates of 3l-64 mg have measured chlorophyll content and
CO2ldm2lh, whereas castor beans may have photosynthetic rate in a wide range of
very similar amounts of chlorophyll (2.4- soybean (Glycine max (L.) Merr.) material,
2.9 mgldm2), but have rates of photosyn- and describe here the relationship we have
thesis of only 18-26 mg
COrldmzlh in observed between these characteristics in
bright light and 300 ppm CO2. Thus the two experiments.
critical differences among species must be
in mesophyll diffusion and kinetics of the
MATERIALS AND METHODS
dark reaction.
Soybean cultivars and strains were planted in a
Gabrielsen (1948) suggested that, to a
field of fertile sandy loam at Harrow, Ontario,
first approximation, light intensity and on the dates shown in Table l. Hills were spaced
chlorophyll content make up a single factor, 0.9 x 0.9 m apart, five or six seeds were planted
namely, light absorption. At high light per hill and the seedlings were thinned to one
intensity, even a small concentration of (cultivar experiment) or two (mutants experi-
chlorophyll can absorb enough light energy ment) plants after emergence. Weeds were
Table l. Dates of planting, conditions at time of measurements and mean values of photosynthetic rate (PJ and
chlorophyll content for the four check cultivars
Pa measured at: Means of 4 check cultivars
Max. CO, PA
Age temp. conc. (mg COr/dm'z/h) Chlorophyll
Planting date (days) CC) (ppm) in full sun) (mg/dm')
Cultivar test
(1) 9 July 1973 50 34 348 34.2 4.76
5l 32 348
(2) 31 May 1974 4t 23 309 33.6 4.42
42 26 309
C hlor ophy ll mutants te st
(1)31May 1974 47 26 309 35.3 3.52
48 30 309
(2)1lJune1975 49 3l st I 34.5 3.40
BUTTERY AND BUZZELL-PHOTOSYNTHESIS IN SOYBEANS
controlled by cultivation and plots were irrigated the l7o level (r : 0.67). Similarly, the two
as necessary. tests of chlorophyll mutants were combined
The mutants experiment (four replications for statistical analysis; differences between
each year) contained 20 lines of soybeans with entries in both P6 and chlorophyll content
various genetical deficiencies in chlorophyll
were established. Pa was closely correlated
with chlorophyll content (r : 0.90).
(seed from the Genetic Type Collection kindly
supplied by Dr. R. Bernard, Soybean Regional
Laboratory, Urbana, Illinois), plus four check The data for the two experiments have
cultivars (Altona, Amsoy, Hawkeye 63 and been plotted in Fig. lA, along with the
Merit). The cultivar experiment (six replications regression lines, and there is very little
each year) contained 48 entries which consisted overlap between the two sets of data. The
Can. J. Plant Sci. Downloaded from cdnsciencepub.com by 49.34.71.181 on 12/26/23
of a wide range of North American commercial
cultivars and experimental lines and included the
same four check varieties as the other experi-
ment.
Rates of photosynthesis were measured on the e
youngest, fully expanded leaves in the field o
using an apparatus modified from that of N
Shimshi (1969). Plants were in the flowering @
stage, 4l-52 days from planting (Table 1).
Radioactive CO2 was supplied for 20 sec to a o-o
small area of leaf enclosed in a plexiglass leaf
chamber exposed to full sunlight. The total COz
concentration of the gas supply is given in Table
l; 1aCO2 content was 3-57o of total. The treated c
o
area was then punched out and dropped into 0.5 =
ml Nuclear-Chicago Solubilizer in a scintillation o
vial. After bleaching with I ml of l27o benzoyl
.a
peroxide, and adding l5 ml of toluene contain- o
ing 8Vo of 2,4-diphenyloxazole, counts were
made on a Beckman LS 250 liquid scintillation
counter. Counting efficiency was determined
from counts before and after adding a measured
volume of labelled hexadecane. Based on the c
o
composition of the gas, the counts were
converted to rate of photosynthesis Pa (:mg o
COzldmzlh) and Pc (:mg CO2/mg
chlorophyll/h). Further details are given by .=
Buttery and Buzzell (1976). The principles of -o
the technique are discussed by Voznesenskii et
al. (1971). o'
Leaf discs were punched out of the side CHLOROPHYLL relotive to Altono
leaflets of the leaves used for phototsynthesis Fig. l.
Relationship between chlorophyll con-
measurements, stored at -15 C for l-3 mo, tent and rate of photosynthesis in soybean
grdund with sand and extracted with 807o leaves. A. Photosynthetic rate versus
acetone for chlorophyll estimation by the chlorophyll content, both on a unit leaf area
method of Arnon (1949). basis. Chlorophyll mutants as open circles;
cultivars as solid black circles. B. Photosynthe-
RESULTS
tic rate per unit leaf area and chlorophyll content
The two soybean cultivar tests gave similar expressed relative.to the check cultivar Altona
results and were combined for statistical which was included in both experiments. C.
analysis. Cultivars differed in both Pa and Photosynthetic rate per unit of chlorophyll and
in chlorophyll content, and these two leaf cholorophyll content both expressed relative
characteristics were positively correlated at to Altona.
CANADIAN JOURNAL OF PLANT SCIENCE
chlorophyll contents were higher for the RuDP carboxylase activity, and mesophyll
four check cultivars in the cultivar test than cell size (Evans 1973), as well as
in the mutants test (Table l). chlorophyll content (Ojima 197 4; W atanabe
In order to put the data from the two 1973) and chloroplast numbers (Kariya and
experiments on the same basis, the chloro- Tsunoda 1972).
phyll and Pa data were transformed relative Using soybean cultivars and lines grow-
to the values of cv. Altona which is ing in the field, and with photosynthetic rate
common to both experiments (Fig. lB); a measured under full sunlight, we have
second-degree polynomial covering both demonstrated a close association between
sets ofdata accounted for nearly gOVo of the Pa and chlorophyll content. This associa-
Can. J. Plant Sci. Downloaded from cdnsciencepub.com by 49.34.71.181 on 12/26/23
variation. tion has been amply confirmed in a number
In the combined analysis of the 48 of smaller tests (unpublished results). The
cultivars, no significant differences in P" simplest explanation for our results is thht
were found, although in the data of I yr, there is a direct causative relationship
analyzed on its own, differences in P" were between quantity of chlorophyll and rate of
established at the 57o level. In the mutants CO2 assimilation: that under field condi-
test, differences in P" were observed, tions chlorophyll content limits Pa. A
significant at the lVo level, with higher slightly modified explanation would be that
values of P" associated with lower an enzyme system closely associated with
chlorophyll contents. A straight regression the chloroplasts controls CO2 uptake. It is
line (P" : 20.6t - 3.41C, where C : mg also possible that chlorophyll content is
chlorophyll/dm2) accounted for 46Vo of thl only related indirectly to Pa, and that some
variation. Transformation of the data from other characteristic is controlling both.
both experiments relative to Altona (Fig. In view of the diverse characteristics of
lC) allowed the fitting of a common leaves described in the literature as being
regression line which accounted for 637o of
correlated with P4, it seems likely that a
the variation.
number of leaf factors may be at or near a
limiting level, and that variation in any one
DISCUSSION of these may cause a corresponding change
Photosynthetic rate estimated from the in P6. Possibly the importance of any
uptake of radioactive CO2 probably repre- individual factor will vary depending on
sents gross rather than net carbon exchange, plant age and nutritional status as well as
since the products of CO2 assimilation do species. Dornhoff and Shibles (1976) con-
not normally appear to be photorespired for cluded that characteristics internal to the
several minutes after uptake (Voznesenskii cells, as opposed to resistances related to
et al. 1971). No differences among soybean stomata, intercellular spaces or cell sur-
cultivars in terms of photorespiration have faces, were regulating PA in soybean
been detected (Ogren and Rinne 1973). leaves. Our results suggest that the quantity
Thus, our results should be closely related of chlorophyll could be one of these internal
to net carbon exchange rate. In comparing cell characteristics.
our values of Pa with the net photosynthetic The results of the cultivar experiment
rates of Dornhoff and Shibles (1970), we showed that 447o of the variability in Pa
found a correlation coefficient of *0.79 for was due to variation in chlorophyll content.
the 16 cultivars common to both sets of This suggests that the initial screening of
data. progenies for high photosynthetic rate could
Varietal differences in photosynthetic be done by measuring chlorophyll content,
rate have been associated with a number of especially if a rapid technique can be
different factors in different crops, includ- developed (such as that of Macnicol et al.
ing stomatal and mesophyll resistance, 1976). Then, having reduced population
BUTTERY AND BUZZELL-PHOTOSYNTHESIS IN SOYBEANS
size, photosynthetic rate could be measured HESKETH, J. D. 1963. Limitations to photo-
more precisely on the selections. synthesis responsible for differences among
species. Crop Sci. 3: 493-496.
ACKNOWLEDGMENTS KARIYA, K. and TSUNODA, S. 1972. Rela-
tionship of chlorophyll content, chloroplast area
We are indebted to the Soya-Bean Growers'
index and leaf photosynthesis rate in Brassica.
Marketing Board for provision of a technician
Tohoku J. Agric. Res. 23: 1-14.
who assisted with the field and laboratory
L. and.
measurements.
MACNICOL, P. K., DUDZNSKI, M.
CONDON, B. N. 1976. Estimation of
ARNON, D. I. 1949. Copper enzymes in chlorophyll in tobacco leaves by direct photo-
isolated chloroplasts. Polyphenoloxidases in metry. Ann. Bot.40: 143-152.
W. L. and RINNE, R. W.
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