the relationship between chlorophyll content and rate ...

CANADAN JOURML OF PI.AI\IT SCIENCE Vol.

57

No. I

January 1977

Can. J. Plant Sci. 1977.57:1-5. Downloaded from www.nrcresearchpress.com by 47.8.106.65 on 06/13/18. For personal use only.

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

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 opintent of leaves and photosynthetic rate has ions. Haberlandt (1914) showed a close relationship between photosynthetic activity Can. J. Plant Sci.57: l-5 (Jan. 1977) I

CANADIAN JOURNAL OF PLANT SCIENCE

and numbers of chloroplasts per unit of leaf

area. However, Willsthtter and Stoll (1918) showed that yellow cultivars of Ulmus and Sambucus could assimilate at rates similar


to those of green varieties of the

same

species. Benedict (1972) calculated that low-chlorophyll mutants of several flowering plants (cotton, pea, peanut, soybean,

(incident quanta) for light to be saturating; it is only at low light levels that the extra light absorbed by a high chlorophyll content becomes important. The results of Sesftik (1966) do not support this hypothesis; his results indicate that the correlation between

rate of photosynthesis and chlorophyll

content was higher at high irradiance than at

and tobacco) have photosynthetic rates per

low.

types.

even individual leaves of many plants are not fully saturated in full sunlight (e.g. Hesketh (1963) for maize; Bowes et al.

milligram of chlorophyll that were 2-ll times higher than that of the normal green

Hesketh (1963) showed that species can

vary greatly in rate of photosynthesis

and

that this variation is not related

to

chlorophyll content. Maize leaves may contain 2.1-3.0 mg chlorophyll/dmz and have photosynthetic rates of 3l-64 mg CO2ldm2lh, whereas castor beans may have very similar amounts of chlorophyll (2.42.9 mgldm2), but have rates of photosyn-

thesis

of only 18-26 mg

COrldmzlh in bright light and 300 ppm CO2. Thus the critical differences among species must be in mesophyll diffusion and kinetics of the dark reaction.

Gabrielsen (1948) suggested that,

first

approximation,

light intensity

to

a

and

chlorophyll content make up a single factor,

namely, light absorption. At high light intensity, even a small concentration of chlorophyll can absorb enough light energy Table

l.

Many crops are never light-saturated;

for soybeans). Thus one could suppose that a higher level of chlorophyll content could further increase light absorption and, therefore, photosynthetic rate. We (1972)

have measured chlorophyll content and photosynthetic rate in a wide range of soybean (Glycine max (L.) Merr.) material, and describe here the relationship we have observed between these characteristics in two experiments.

MATERIALS AND METHODS Soybean cultivars and strains were planted in a field of fertile sandy loam at Harrow, Ontario, on the dates shown in Table l. Hills were spaced 0.9 x 0.9 m apart, five or six seeds were planted per hill and the seedlings were thinned to one (cultivar experiment) or two (mutants experiment) plants after emergence. Weeds were

Dates of planting, conditions at time of measurements and mean values of photosynthetic rate chlorophyll content for the four check cultivars Means of 4 check cultivars

Pa measured at:

Planting date

Age (days)

Max. temp. CC)

CO,

PA

COr/dm'z/h) in full sun)

(mg

conc. (ppm)

Chlorophyll (mg/dm')

Cultivar test

(1)

9 July 1973

(2) 31 May 1974

50 5l 4t 42

348

34.2

4.76

23 26

348 309 309

33.6

4.42

26 30

309 309

35.3

3.52

3l

st I

34.5

3.40

34 32

C hlor ophy

(1)31May 1974 (2)1lJune1975

47 48 49

(PJ

ll mutants te st

and

BUTTERY AND BUZZELL-PHOTOSYNTHESIS

(r

:

0.67). Similarly, the two

controlled by cultivation and plots were irrigated

the l7o level

as necessary.

tests of chlorophyll mutants were combined for statistical analysis; differences between

The mutants experiment (four replications each year) contained 20 lines of soybeans with


IN SOYBEANS

various genetical deficiencies in chlorophyll (seed from the Genetic Type Collection kindly supplied by Dr. R. Bernard, Soybean Regional Laboratory, Urbana, Illinois), plus four check cultivars (Altona, Amsoy, Hawkeye 63 and Merit). The cultivar experiment (six replications each year) contained 48 entries which consisted of a wide range of North American commercial cultivars and experimental lines and included the same four check varieties as the other experiment. Rates of photosynthesis were measured on the

youngest, fully expanded leaves

in the field

using an apparatus modified from that of Shimshi (1969). Plants were in the flowering stage, 4l-52 days from planting (Table 1). Radioactive CO2 was supplied for 20 sec to a 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 area was then punched out and dropped into 0.5 ml Nuclear-Chicago Solubilizer in a scintillation vial. After bleaching with I ml of l27o benzoyl peroxide, and adding l5 ml of toluene containing 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

of the gas, the

counts were converted to rate of photosynthesis Pa (:mg composition

COzldmzlh) and Pc (:mg

CO2/mg chlorophyll/h). Further details are given by Buttery and Buzzell (1976). The principles of the technique are discussed by Voznesenskii et al. (1971). Leaf discs were punched out of the side leaflets of the leaves used for phototsynthesis measurements, stored at -15 C for l-3 mo,

grdund with sand and extracted with acetone

807o

for chlorophyll estimation by

entries in both P6 and chlorophyll content were established. Pa was closely correlated with chlorophyll content (r : 0.90).

The data for the two experiments have been plotted in Fig. lA, along with the regression lines, and there is very little overlap between the two sets of data. The

e

o

N

@

o-o

c

o

= o .a

o

c o o .= -o

o' CHLOROPHYLL

Fig.

relotive to Altono

l.

Relationship between chlorophyll conof photosynthesis in soybean leaves. A. Photosynthetic rate versus

tent and rate

method of Arnon (1949).

chlorophyll content, both on a unit leaf area basis. Chlorophyll mutants as open circles;

RESULTS The two soybean cultivar tests gave similar results and were combined for statistical analysis. Cultivars differed in both Pa and in chlorophyll content, and these two characteristics were positively correlated at

cultivars as solid black circles. B. Photosynthetic rate per unit leaf area and chlorophyll content expressed relative.to the check cultivar Altona which was included in both experiments. C. Photosynthetic rate per unit of chlorophyll and leaf cholorophyll content both expressed relative to Altona.

the

CANADIAN JOURNAL OF PLANT SCIENCE

chlorophyll contents were higher for the four check cultivars in the cultivar test than in the mutants test (Table

l).

In order to put the data from the two experiments on the same basis, the chlorophyll and Pa data were transformed relative


to the values of cv. Altona which

common to both experiments (Fig.

is

lB);

a

second-degree polynomial covering both sets ofdata accounted for nearly gOVo of the variation. In the combined analysis of the 48

cultivars, no significant differences in P" were found, although in the data of I yr, analyzed on its own, differences in P" were established at the 57o level. In the mutants test, differences in P" were observed, significant at the lVo level, with higher values of P" associated with lower chlorophyll contents. A straight regression

line (P" : 20.6t - 3.41C, where C : mg chlorophyll/dm2) accounted for 46Vo of thl variation. Transformation of the data from both experiments relative to Altona (Fig.

lC) allowed the fitting of a common regression line which accounted for 637o of the variation.

DISCUSSION Photosynthetic rate estimated from the uptake of radioactive CO2 probably represents gross rather than net carbon exchange,

since the products of CO2 assimilation do not normally appear to be photorespired for several minutes after uptake (Voznesenskii et al. 1971). No differences among soybean cultivars in terms of photorespiration have

been detected (Ogren and Rinne 1973). Thus, our results should be closely related to net carbon exchange rate. In comparing our values of Pa with the net photosynthetic rates of Dornhoff and Shibles (1970), we found a correlation coefficient of *0.79 for the 16 cultivars common to both sets of data.

Varietal differences in photosynthetic rate have been associated with a number of different factors in different crops, includ-

ing

stomatal and mesophyll resistance,

RuDP carboxylase activity, and mesophyll

cell size (Evans 1973), as well

as

chlorophyll content (Ojima 197 4; W atanabe 1973) and chloroplast numbers (Kariya and Tsunoda 1972).

Using soybean cultivars and lines growing in the field, and with photosynthetic rate measured under

full

sunlight, we have

demonstrated a close association between

Pa and chlorophyll content. This association has been amply confirmed in a number

of smaller tests (unpublished results). The simplest explanation for our results is thht there is a direct causative relationship between quantity of chlorophyll and rate of CO2 assimilation: that under field conditions chlorophyll content limits Pa. A slightly modified explanation would be that an enzyme system closely associated with the chloroplasts controls CO2 uptake. It is

also possible that chlorophyll content is only related indirectly to Pa, and that some other characteristic is controlling both. In view of the diverse characteristics of leaves described in the literature as being correlated with P4, it seems likely that a number of leaf factors may be at or near a limiting level, and that variation in any one

of these may cause a corresponding change in P6. Possibly the importance of any

individual factor will vary depending on

plant age and nutritional status as well as species. Dornhoff and Shibles (1976) concluded that characteristics internal to the cells, as opposed to resistances related to stomata, intercellular spaces or cell sur-

faces, were regulating PA

in

soybean

leaves. Our results suggest that the quantity of chlorophyll could be one of these internal cell characteristics. The results of the cultivar experiment showed that 447o of the variability in Pa was due to variation in chlorophyll content. This suggests that the initial screening of progenies for high photosynthetic rate could be done by measuring chlorophyll content,

especially

if a rapid

technique can be

developed (such as that of Macnicol et al. 1976). Then, having reduced population

BUTTERY AND BUZZELL-PHOTOSYNTHESIS

size, photosynthetic rate could be measured more precisely on the selections.

HESKETH, J. D. 1963. Limitations to photosynthesis responsible for differences among

ACKNOWLEDGMENTS

KARIYA, K. and TSUNODA, S. 1972. Rela-

We are indebted to the Soya-Bean Growers' Marketing Board for provision of a technician

who assisted with the field and


IN SOYBEANS

laboratory

measurements.

D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidases in Beta vulgaris . Plant Physiol. 24: l-15 . BENEDICT, C. R. 19'72. Net COz fixation in ARNON,

yellow-green plants. Pages 7-25 in C. C. Black, ed. Net carbon dioxide assimilation in higher plants. Symp. Sect. Amer. Soc. Plant Physiol. BOWES, G., OGREN, W. L. and HAGEMAN, R. H. 19'72. Light saturation, photosynthesis rate, RuDP carboxylase activity, and specific leaf weight in soybeans grown under different light intensities. Crop. Sci. 12:7'l-79.

BUTTERY, B. R. and BUZZELL, R.

I.

1976.

Flavonol glycoside.genes and photosynthesis in soybeans. Crop. Sci. 16:547-55O. DORNHOFF, G. M. and SHIBLES, R. M. 1970. Varietal differences in net photosynthesis ofsoybean leaves. Crop. Sci. l0: 42-45. DORNHOFF, G. M. and SHIBLES, R. M. 1976. Leaf morphology and anatomy in relation to CO2-exchange rate of soybean leaves. Crop

Sci.16:377-381. EVANS, L. T. 1973. The effect of light on plant growth, development and yield. Pages 2l-35 in R. O. Slatyer, ed. Plant response to climatic factors. UNESCO, Paris. GABRIELSEN, E. K. 1948. Effects of different chlorophyll concentrations on photosynthesis in foliage leaves. Physiol. Plant. l:5-37. HABERLANDT, G. 1914. Physiological plant

anatomy.

(M.

Drummond, transl.), 3rd ed.

Macmillan, London.

species. Crop Sci. 3: 493-496.

tionship of chlorophyll content, chloroplast area index and leaf photosynthesis rate in Brassica. Tohoku J. Agric. Res. 23: 1-14.

L. and. CONDON, B. N. 1976. Estimation of chlorophyll in tobacco leaves by direct photoMACNICOL, P. K., DUDZNSKI, M. metry. Ann. Bot.40: 143-152.

OGREN,

W. L. and RINNE, R. W.

1973.

Photosynthesis and seed metabolism. Pages

391-416

in B. E.

Caldwell, ed.

Soybeans:

improvement, production and uses. Amer. Soc. Agron. lnc. Madison, Wis. OJIMA, M. lg74.Improvement of photosynthetic capacity in soybean variety. Jap. Agric. Res. Q. 8: 6-12.

SESTAK, Z. 196;6. Limitations for finding a linear relationship between chlorophyll content and photosynthetic activity. Biol. Plant (Praha) 8:336-346. SHIMSHI, D. 1969. A rapid field method for measuring photosynthesis with labelled carbon dioxide. J. Exp. Bot.20:381-390.

VOZNESENSKII, V. L., ZALENSKII, O. V. and AUSTIN, R. B. 1971. Methods of measuring rates of photosynthesis using carbon-14 dioxide. Pages 276-293 in Z. Sestak, J. Catsky

and P. G. Jarvis, eds. Plant photosynthetic

production, manual of methods. Dr. W. Junk,

N.V. Publishers, The Hague, 1971. WATANABE, l. 1973. Mechanism of varietal

differences in photosynthetic rate of soybean leaves. l. Correlations between photosynthetic rates and some chloroplast characters. Proc. Crop Sci. Soc. Jap. 43:377-386. WILLSTATTER, R. ANd STOLL, A. I9I8.

Untersuchungen

iiber die assimilation

kohlenshure. Verlag Springer, Berlin.

der