Photosynthesis Research 12:13-23 (1987) © Martinus Nijhoff Publishers, Dordrecht--Printed in the Netherlands
13
Regular paper
Determination of the total chlorophyll distribution pattern in living leaves HARALD BOLHAR-NORDENKAMPF & ELISABETH GRONWEIS Institut ffir Pflanzenphysiologie, Universitgtt Wien, A-1091 Vienna, Althanstrafle 14, Austria Received 3 March 1986; accepted in revised form 26 August 1986 Key words: Tradescantia albiflora Briickn., Phaseolus vulgar& L., atrazine, bleaching effects, chlorophyll content determination, iron deficiency,living tissue Abstract. Tradescantia albiflora-leaves were used in developing a determination method for chlorophyll in living leaves using a microscopic spectro photometer (MPV, Leitz). The wavelength of the maximal absorption for chlorophyll a was found to be at 670 nm and for chlorophyll b at 652 rim. To calculate the reference values the intensity of the transmitted light at 750 nm was measured. The absorption at 750 nm results only from the chlorophyll free structure in the leaves. To correct optical errors the two-wavelength method was used. The values gained in arbitrary units were calibrated against data measured in the conventional way. The resulting calibration line shows a very high correlation coefficientwhere r2 = 0.997. It was proved that the calibration line was also correct for determinations with leaves from other plant species. Using this determination method the chlorophyll content of small areas on the living leaf blade of Phaseolus vulgaris was determined. As consequence of the thicker mesophyll accompanying the veins the chlorophyll concentration is 30% higher in this tissue. A lower chlorophyll concentration was observed in the thinner leaf tip and in the oldest regions at the leaf ground. At the leaf tip, the leaf ground and in the tissue along the veins an iron deficitcauses less bleaching than in the areoles. In the same leaf regions the bleaching herbicide atrazine induces rapid bleaching if supplied through transpiration stream. The application of Atrazine on the leaf surface gives rise to the contrary effect. All these phenomena seem to be a result of a differing water supply by the xylem vessels.
Introduction
T h e d e t e r m i n a t i o n o f the chlorophyll c o n t e n t of leaves is a key technique in studies of photosynthesis a n d for e s t i m a t i o n of b i o p r o d u c t i v i t y ( M e d i n a a n d Lieth, 1964). The absolute a m o u n t of chlorophyll a n d the changes in concentration over a period of time led to conclusions a b o u t the m i n e r a l n u t r i e n t supply as well as a b o u t the m o d e of action o f herbicides. T h e m e t h o d n o r m a l l y used to determine chlorophyll can be destructive. The chloroplast p i g m e n t s are extracted into organic solvents from a k n o w n a m o u n t of leaf material a n d c o n c e n t r a t i o n s are d e t e r m i n e d spectrophotometrically (Ziegler a n d Egle, 1965a, b; Inskep a n d Bloom, 1985). U s i n g this m e t h o d small areas ( < 1 m m -2) with differing chlorophyll c o n t e n t c a n n o t be easily separated a n d n o material is left for sequential investigations. Therefore the object was to develop a n in vivo n o n - d e s t r u c t i v e m e t h o d for m e a s u r e m e n t of the chlorophyll c o n c e n t r a t i o n in small spots of the leaf.
14 This method was tested to examine whether the chlorophyll concentration in a leaf, which looks homogeneously green to the unaided eye, shows a distribution pattern on the leaf lamina (Nagel, 1940; Hardcare et al., 1984). This would reveal any changes in distribution pattern after treatments known to cause deficiencies in chlorophyll content. These investigations should also reveal, how rapidly a certain treatment causes a detectable reduction in chlorophyll content.
Material and methods
Chlorophyll concentration was measured with a microscopic spectrophotometer (MPV I, Leitz), mounted on a research microscope (Ortholux, Leitz). This instrument enables absorption measurements even of small regions in microscopic slides. The measuring diaphragm together with lenses of high magnification enables absorption measurements in spots with a diameter down to 4/~m2. To illuminate the preparation low voltage light source with a stabilized power supply is employed. The wavelength of the measuring beam is, adjusted by means of an interference graduated line filter, continuously variable (Veril S 200, Schott). A special lens below the microscopic table is used to focus the field diaphragm in the plane of the object. To avoid errors of the field margins only two thirds in the center of the illuminated area are used for the entire measurement. To minimize the influence from light scattering, lenses with high numeric aperture were used during the measurements. Using the lowest magnification an area of 0.024 mm 2 on the leaf surface was measured. The transmitted light is received by a photomultiplier (RCA 1 P 21), supplied with a stabilized high voltage unit. The resulting current is indicated by a reflecting galvanometer or by a recorder. The reference measurements were made on a common double beam spectrophotometer (Acta C III, Beckmann). In developing the method relatively thin leaves with good infiltration properties were required. Tradescantia albiflora (Br/ickn.) fits these criteria and, in addition, is easy to propagate as a clone. There is also a variegated leaf variety with large chlorophyll free areas (cv. Albo-vittata) which were intended to be used as a reference measuring point. The plants were cultivated in a controlled environment with day/night temperature of 2 ~ 1 8 °C and a photon flux density of 35 #tool. m 2. s-l equivalent to 9 W . m -2. To investigate the bleaching phenomena bush beans (Phaseolus vulgaris var. nanus L. 'Sabo Processor') were cultivated in a complete nutrient solution enriched with trace elements. The chlorophyll deficiencies were induced by keeping them in the dark by cultivation in an iron deficient solution
15 by cultivation in a complete nutrient solution containing the bleaching herbicide atrazine and by applying atrazine to the leaves. Middle leaflets of the first and second trifoliate leaf were used for the measurements. The measuring points on the leaf lamina were set according to schemes, called profiles (Fig. 3). These profiles take into consideration both the areoles and the areas near the thicker veins. When drawing the figures the mean of the values along the longitudinal profiles was used as a reference magnitude.
Measuring method Chlorophyll concentrations are determined in solutions from the light extinction at specific wavelengths. To measure the chlorophyll concentration in a n intact leaf one has to consider chlorophyll as if it were dissolved in the leaf structure. In addition the assumption has to be made that it is correct to measure chlorophyll concentrations in a multicompound solution using the extinction data at specific wavelengths. If the chlorophyll concentration is determined in a solution (I~) the pure solvent is used as the reference (I0). For the measurements on a living leaf a suitable reference has to be found. The reference should represent all absorbing structures on a leaf besides the chlorophyll. This should be in fact at a wavelength where chlorophyll is not absorbing any more whereas the leaf structure shows unchanged absorption. To establish this absorption curves of the leaf tissue have to be measured. For reference the following two should be used: An object free spot in the preparation and a leaf tissue free from chlorophyll. The first attempt used the white part of a variegated leaf as a reference (Fig. 1). The resulting extinction curve shows maximal absorption of light between 669.2 and 671.4 nm. Above 700 nm, where no chlorophyll absorption occurs, a relatively high extinction value is still observed. This means that the chlorophyll-free structures in the green parts of a leaf show a higher absorption in comparison with the white parts. It was found that the white areas are in general 18.5% thinner than the green ones. If leaf pieces were used for reference which were previously bleached by boiling ethanol, zero extinction is measured at 750nm (Fig. 1). As the absorption of both, the green living leaf and the bleached tissue is equal at 750nm, at this wavelength the extinction of a green leaf with an adjacent empty area of the slide for reference values can serve as a standard of comparison. To get the reference values for other wavelengths the transmitted light intensity through a bleached tissue was measured at different wavelengths setting the amount of transmitted light intensity at 750 nm as 1. The decline of absorption from 550 nm to 750 nm is due to the decrease of light scattering with higher wavelength (Goldstein, 1970). As a result of several series of measurements with bleached tissues of different leaves it was proved that the relations (k~) between the transmitted light at
16
669,2 - 671,z~nm 1,5
,.'
:,
oJ
1,0 ,,.,
, .,.'''
•
I
,,.'"
I
: =:::-: :- : : 2 _
.-'"
..
'
~
•
...... :.---..
0,5
550
600
650
700 wQvelenght[nm]
%0
Fig. 1. Extinction of green Tradescantia leaves measured with a microscopic spectrophotometer: - - with artificially bleached leaf tissue for reference, - - with a white part of variegated leaf for reference, . .... with an object free area for reference, - - - , extinction of an artificially bleached leaf tissue with an object free area for reference.
750 nm and those at other wavelengths remain constant. These relations also take in consideration light scattering and differences in the spectral sensitivity o f the whole measuring device, mainly represented by the spectrum o f the measuring light and the photomultiplier.
I~(,~)
k~ -
•,(750)
-
const.
The wavelength specific factor k~ is used to calculate absorption o f leafstructure at a selected wavelength using the absorption value at 750 n m only. Therefore a bleached leaf tissue is not needed any longer to d e t e r m i n e I0 at any wavelength, 10(2)
=
11(750) " k ~ .
Selection of the measuring wavelengths In c h l o r o p h y l l estimation the extinctions are determined at the wavelengths o f m a x i m a l absorption for c h l o r o p h y l l a a n d b. The wavelengths o f these m a x i m a v a r y a c c o r d i n g to the solvent. T h e wavelength for m a x i m u m extinction o f c h l o r o p h y l l a in the living green
17 leaf tissue is determined at 670nm (see French et al., 1972). The respective wavelength for chlorophyll b is marked by a hardly detectable shoulder in the absorption curve. As a chlorophyll a solution in 15% bovine serum albumine shows the extinction maximum at 670 nm, clorophyll b was dissolved as well in BSA. The maximum extinction was found at 652 nm (see Demeter et al., 1976; Tombacz et a l., 1984). The position of the wavelength with half maximal extinction was found 15 nm above the maximum for chlorophyll a.
The two-wavelength method to correct cytophotometric errors in measurement
The errors involved in the microspectrophotometrical method are light scatter, distributional error and errors due to the spectrophotometer. By comparison of the methods of duplicated estimates, two-wavelength photometry minimises these errors (Patai, 1952; Ornstein, 1957; Garcia, 1962 a, b). 1-
7 =
1 -
1 -~ ~Fm -
2ThJ 1OgTm _
Tm- Th Tm -
T~
1 + Tm-2T h = corrected extinction value; Tm = transmittance at the wavelength with the maximal absorption (670 nm); Th = transmittance at the wavelength with half maximal absorption (685 nm and 652 nm). In theory the two-wavelength method is suitable only for solutions containing one pigment (Patzelt, 1969). However, it has been found that the corrected extinction value can be calibrated against data gained from comparable leaf material measured in conventional spectrophotometer. In this procedure the area of the photometric field is not needed for calculations. Nevertheless the photometric field should be an area with homogenous structure. The size and shape of the measuring diaphragm should be selected according to the tissue under study and should not be changed during a series of measurements. The transmittance was determined at 670 nm, 685 nm and 652 nm. The transmittances at 652nm and 685nm were used both in the calculations as Th. Therefore two corrected extinction values were recieved. The mean value of these two was used to find the corresponding chlorophyll concentrations on the calibration line (Fig. 2). The aim of this procedure was to take sufficient account of the chlorophyll b content.
The construction o f a calibration line
As stated previously the data calculated from measurements with the MPV were compared with those gained with a conventional spectrophotometer. For this
18
0,05 ff E
0,04
I *) 0,03
0,02
0,01
I
O,S
t,0
i
I
i
1,5 CORRECTED EXT. VALUES
Fig. 2. Calibration line to obtain absolute chlorophyll concentrations using values derived from living leaf in the microscopic spectralphotometer.
a
purpose about 100 measurements were taken with the MPV in a single sample. After this the total leaf area of the sample was determined and the chlorophyll was quantitatively extracted. To get different chlorophyll content in the Tradescantia leaves used, bleaching was induced by high light intensity and by the application of the herbicide atrazine. The resulting calibration line follows the equation y
=
0.0276x - 0.0006.
The correlation coefficient reaches r2 = 0.997.
19
Fig. 3. Arrangement of the measuring profiles on the leaf blade: L = longitudinal profile, C = cross profile, Dr, D L = diagonal profiles (right and left side).
To test the reliability of the equation found, leaves from several other plants were examined (Zea mays, Abutilon sp., Acalypha sp., Phaseolus vulgaris). The data received are in good agreement with the calibration line (Fig. 2). Even from leaves with a high or low ratio of chlorophyll a to b the data gained show a good correlation.
Results
The chlorophyll distribution pattern of bean leaves Plants in a complete nutrient solution (controls)
The unaided eye is not able to detect differences in the chlorophyll content of various areas of the leaf lamina of first and second trifoliate leaves. Using the MPV the highest chlorophyll concentrations (0.031 mg. cm 2) were determined in the apical part of the lamina. The leaf tip and leaf base showed values which were 5-10% and 10-15% lower, respectively. Along the diagonal profiles the chlorophyll concentration in the thickened tissue near the veins was 20-30% higher in comparison with the areoles (Fig. 4).
Plants cultivated in the dark
After 17 days under favourable conditions in the green house the plants were transferred to the dark for one week. In comparison with the controls this
20 treatment causes on average a 20% reduction in the chlorophyll concentration throughout the lamina. In single leaves the distribution pattern of the chlorophyll content varied. Some leaves showed no change in concentration at the leaf tip, others underwent a 5% higher deficit in the tissue near the veins than in the areoles.
Plants grown in an iron deficient nutrient solution
After 14 to 21 days characteristic iron deficiency symptoms will develop in plants on iron deficient media (Machold and Stephan, 1969). The second trifoliate leaves develop bleached areoles with green tissue accompanying the veins (Terry and Low, 1982). In comparison with the controls the chlorophyll concentration is generally 60% lower along the longitudinal profil. An exception is, that chlorophyll content at the leaf tip is only 30% lower. Along the diagonal profile the chlorophyll reduction increases from the central vein to the leaf margin by 20%. In the tissue along the veins the chlorophyll loss is less marked. This difference in concentration between the areoles and the tissue near the venation is three times greater as observed in the controls (Fig. 5).
rlv 120
2
i}
_ /.-/"
1O0
De
/" L °"
Dr
Fig. 4. Chlorophyll concentration in mg. cm -2 leaf area, first trifoliate leaves: . . . . . . L, nv = tissue near the veins Dr, D1, a = areoles - - - C (mid vein) 100% = 0.0293 rag. cm -2 and equals the mean of all values measured along L.
100
nv B0
60
40
n G ii
L
,I
a
/
li
De
Dr
Fig. 5. Iron deficit: Chlorophyll concentration in percent of the controls (see Fig. 4).
21
Plants treated with the herbicide Atrazine Atrazine (2-chloro-4-ethylamine-6-isopropyl-amino-s-triazine) is absorbed by leaves and roots. A concentration of 10-Smol. 1 ~ in the apoplast solution causes severe bleaching dependent on temperature and irradiation (BolharNordenkampf, 1975).
Application of Atrazine on the leaf lamina A solution containing 500 ppm atrazine (Gesaprim, Ciba-Geigy) was applied to one half of the leaf. Two days later the different profiles of each half were measured. The treated half develops an all-over chlorophyll deficit from 5-25 % with the highest losses in the areoles. The leaf tip is not influenced. The bleaching effect is restricted exclusively to the treated half of the lamina.
Application of Atrazine via the transpiration stream The plants were grown for 5 days in a complete nutrient culture solution containing 100 ppm Atrazine. After one day a chlorophyll loss of 5% became detectable at the leaf tip. Subtoxic atrazine concentrations in other parts of the lamina cause an increase in the chlorophyll content of 20% (see Krakkai, 1973). Chlorophyll concentration in the areoles does not become lower than in the controls before 5 days. In contrast the chlorphyll content of the tissue near the veins drops continuously from the second day and reaches values 10 to 20% below those of the areoles (Fig. 6).
Discussion
The enhanced chlorophyll concentration in the tissue near the vein is determined by the leaf anatomy. Along the veins the spongy parenchyma is constantly 30% thicker because of two additional cell layers. Although the minor veins near the leaf margin are considerably thinner than the main veins, the chlorophyll absorption in the accompanying parenchymatous tissue varies little. The lower chlorophyll concentration on the leaf tip is determined by a thinner
120t
--] i
a
1001 De
nv Ii
L'
--L. " --tJ G
',', Dr
Fig. 6. Complete nutrient solution containing 1000ppm Atrazine 2 days after adding herbicide (see
22 c h l o r e n c h y m a , at the leaf base the same p h e n o m e n is the result o f a c h l o r o p h y l l loss caused b y age. I r o n deficit caused a c h a n g e in the p a t t e r n o f c h l o r o p h y l l content, which d e p e n d s on the s u p p l y a n d d i s t r i b u t i o n o f the i r o n still available in the xylem sap. In general the tissue a l o n g the veins, the leaf base a n d tip are supplied better b y the xylem stream a n d therefore in these regions o f the leaf b l a d e the chlorosis is less. In the case o f an atrazine s u p p l y via the nutrient solution the herbicide is d i s t r i b u t e d in the same w a y as the iron ions. This fact leads to r a p i d bleaching on the c h l o r e n c h y m a a d j a c e n t to the veins. The c o n t r a r y effect is observed, when atrazine is a p p l i e d to the surface o f the leaf lamina. In leaf regions with a better w a t e r s u p p l y f r o m the xylem s t r e a m the diffusing herbicide is diluted a n d the bleaching is less. In c o n t r a s t the diffusive stress by placing the p l a n t s in the d a r k causes an overall bleaching with no c o r r e l a t i o n to the leaf a n a t o m y . These results show t h a t the d e v e l o p e d technique is suitable to m e a s u r e small differences in c h l o r o p h y l l d i s t r i b u t i o n on a leaf blade.
References Bolhar-Nordenkampf HR (1975) Die Ver/inderungen des Chlorophyllgehaltes in ontogenetisch verschiedenen Bl~ittern von Phaseolus vulgaris var. nanus L. nach Behandlung mit Atrazin. Biochem Physiol. Pflanzen (BPP) Bd 167:41-64 Demeter S., Sagromsky H., Faludi-Daniel A., (1976): Orientation of chlorophyll b in thylakoids of barley chloroplasts. Photosynthetica 10(2):193-197 French C.S., Brown J.S. and Lawrence M.C., (1972) Four universal forms of chlorophyll a. Plant Physiology 49:421-429 Garcia A.M., (1962) Studies in Leucocytes and related cells of mammals. I. On microspectrophotometric errors and statistical models. Histochemie 3:170-177 Garcia A.M., (1962) II. On the Feulgen reaction and Two-Wavelength microspectrophotometry. Histochemie 3:178 154 Hardacre A.K. Nicholson H.F. and Boyce M.L.P., (1984) A portable photometer for the measurement of chlorophyll in intact leaves. New Zealand Journal of Experimental Agriculture 12:357-362 Inskeep W.P. and Bloom P.R., (1985) Extinction coefficients of chlorophyll a and b in N, Ndimethylformamide and 80% acetone. Plant Physiology 77:483-485 Krakkai I. (I 973) Herbicide derived from chlortriazine as permanent stimulators of maize. Proceedings of the Research Institute of Pomology, Skierniewice, Poland Nr.3, 352-357 Machold O. and Stephan U.W., (1969) The function of iron in porphyrin and chlorophyll biosinthesis. Phytochemistry 8:2189-2192 Medina E. and Lieth H., (1964) Die Beziehungen zwischen Chlorophyllgehalt, assimilierender Flfiche und Trockensubstanzproduktion in einigen Pflanzengemeinschaften. Beitr. Biol. Pflanzen 40:451-494 Nagel W. (1940) Ober die Blattfarbstoffe des Tabak. Bot. Archiv 40:1-57 Ornstein L., (1952) The distributional error in microspectrophotometry. Lab Invest 1:250-265 Patau K., (1952) Absorption microphotometry of irregular shaped objects. Chromosoma 5, Berl. 341-362 Patzelt W.J., (1969) Absorptionsmessungen: Grundlagen und Anwendungen. Mitt.a.Labor Anw.Mikro, Mikrophotometrie, 32 a
23 Terry N and Low G (1982) Leaf chlorophyll content and its relation to the intercellular localization of iron. J Plant Nutrition 5:301-310 Tombacz E., Varonyi Z. and Szalay L., (1983) Artificial chlorophyll-protein complexes. In: Sybesma C (ed) Advances in Photosynthesis Research, Vol. 2. The Hague: Martinus Nijhoff/Dr W. Junk Publishers Ziegler R and Egle K., (1965) Zur quantitativen Analyse der Chloroplastenpigmente. I. Kritische Oberprfifung der spektralphotometrischen Chlorophyll-Bestimmung. Betr Biol Pflanzen 41:11-37 Ziegler R and Egle K. (1965b) II Verfinderungen im Chlorophyllspiegel bei ausdifferenzierten Laubbl/ittern im Laufe eines Tages. Beitr. Biol. Pflanzen 41:39-63
13
Regular paper
Determination of the total chlorophyll distribution pattern in living leaves HARALD BOLHAR-NORDENKAMPF & ELISABETH GRONWEIS Institut ffir Pflanzenphysiologie, Universitgtt Wien, A-1091 Vienna, Althanstrafle 14, Austria Received 3 March 1986; accepted in revised form 26 August 1986 Key words: Tradescantia albiflora Briickn., Phaseolus vulgar& L., atrazine, bleaching effects, chlorophyll content determination, iron deficiency,living tissue Abstract. Tradescantia albiflora-leaves were used in developing a determination method for chlorophyll in living leaves using a microscopic spectro photometer (MPV, Leitz). The wavelength of the maximal absorption for chlorophyll a was found to be at 670 nm and for chlorophyll b at 652 rim. To calculate the reference values the intensity of the transmitted light at 750 nm was measured. The absorption at 750 nm results only from the chlorophyll free structure in the leaves. To correct optical errors the two-wavelength method was used. The values gained in arbitrary units were calibrated against data measured in the conventional way. The resulting calibration line shows a very high correlation coefficientwhere r2 = 0.997. It was proved that the calibration line was also correct for determinations with leaves from other plant species. Using this determination method the chlorophyll content of small areas on the living leaf blade of Phaseolus vulgaris was determined. As consequence of the thicker mesophyll accompanying the veins the chlorophyll concentration is 30% higher in this tissue. A lower chlorophyll concentration was observed in the thinner leaf tip and in the oldest regions at the leaf ground. At the leaf tip, the leaf ground and in the tissue along the veins an iron deficitcauses less bleaching than in the areoles. In the same leaf regions the bleaching herbicide atrazine induces rapid bleaching if supplied through transpiration stream. The application of Atrazine on the leaf surface gives rise to the contrary effect. All these phenomena seem to be a result of a differing water supply by the xylem vessels.
Introduction
T h e d e t e r m i n a t i o n o f the chlorophyll c o n t e n t of leaves is a key technique in studies of photosynthesis a n d for e s t i m a t i o n of b i o p r o d u c t i v i t y ( M e d i n a a n d Lieth, 1964). The absolute a m o u n t of chlorophyll a n d the changes in concentration over a period of time led to conclusions a b o u t the m i n e r a l n u t r i e n t supply as well as a b o u t the m o d e of action o f herbicides. T h e m e t h o d n o r m a l l y used to determine chlorophyll can be destructive. The chloroplast p i g m e n t s are extracted into organic solvents from a k n o w n a m o u n t of leaf material a n d c o n c e n t r a t i o n s are d e t e r m i n e d spectrophotometrically (Ziegler a n d Egle, 1965a, b; Inskep a n d Bloom, 1985). U s i n g this m e t h o d small areas ( < 1 m m -2) with differing chlorophyll c o n t e n t c a n n o t be easily separated a n d n o material is left for sequential investigations. Therefore the object was to develop a n in vivo n o n - d e s t r u c t i v e m e t h o d for m e a s u r e m e n t of the chlorophyll c o n c e n t r a t i o n in small spots of the leaf.
14 This method was tested to examine whether the chlorophyll concentration in a leaf, which looks homogeneously green to the unaided eye, shows a distribution pattern on the leaf lamina (Nagel, 1940; Hardcare et al., 1984). This would reveal any changes in distribution pattern after treatments known to cause deficiencies in chlorophyll content. These investigations should also reveal, how rapidly a certain treatment causes a detectable reduction in chlorophyll content.
Material and methods
Chlorophyll concentration was measured with a microscopic spectrophotometer (MPV I, Leitz), mounted on a research microscope (Ortholux, Leitz). This instrument enables absorption measurements even of small regions in microscopic slides. The measuring diaphragm together with lenses of high magnification enables absorption measurements in spots with a diameter down to 4/~m2. To illuminate the preparation low voltage light source with a stabilized power supply is employed. The wavelength of the measuring beam is, adjusted by means of an interference graduated line filter, continuously variable (Veril S 200, Schott). A special lens below the microscopic table is used to focus the field diaphragm in the plane of the object. To avoid errors of the field margins only two thirds in the center of the illuminated area are used for the entire measurement. To minimize the influence from light scattering, lenses with high numeric aperture were used during the measurements. Using the lowest magnification an area of 0.024 mm 2 on the leaf surface was measured. The transmitted light is received by a photomultiplier (RCA 1 P 21), supplied with a stabilized high voltage unit. The resulting current is indicated by a reflecting galvanometer or by a recorder. The reference measurements were made on a common double beam spectrophotometer (Acta C III, Beckmann). In developing the method relatively thin leaves with good infiltration properties were required. Tradescantia albiflora (Br/ickn.) fits these criteria and, in addition, is easy to propagate as a clone. There is also a variegated leaf variety with large chlorophyll free areas (cv. Albo-vittata) which were intended to be used as a reference measuring point. The plants were cultivated in a controlled environment with day/night temperature of 2 ~ 1 8 °C and a photon flux density of 35 #tool. m 2. s-l equivalent to 9 W . m -2. To investigate the bleaching phenomena bush beans (Phaseolus vulgaris var. nanus L. 'Sabo Processor') were cultivated in a complete nutrient solution enriched with trace elements. The chlorophyll deficiencies were induced by keeping them in the dark by cultivation in an iron deficient solution
15 by cultivation in a complete nutrient solution containing the bleaching herbicide atrazine and by applying atrazine to the leaves. Middle leaflets of the first and second trifoliate leaf were used for the measurements. The measuring points on the leaf lamina were set according to schemes, called profiles (Fig. 3). These profiles take into consideration both the areoles and the areas near the thicker veins. When drawing the figures the mean of the values along the longitudinal profiles was used as a reference magnitude.
Measuring method Chlorophyll concentrations are determined in solutions from the light extinction at specific wavelengths. To measure the chlorophyll concentration in a n intact leaf one has to consider chlorophyll as if it were dissolved in the leaf structure. In addition the assumption has to be made that it is correct to measure chlorophyll concentrations in a multicompound solution using the extinction data at specific wavelengths. If the chlorophyll concentration is determined in a solution (I~) the pure solvent is used as the reference (I0). For the measurements on a living leaf a suitable reference has to be found. The reference should represent all absorbing structures on a leaf besides the chlorophyll. This should be in fact at a wavelength where chlorophyll is not absorbing any more whereas the leaf structure shows unchanged absorption. To establish this absorption curves of the leaf tissue have to be measured. For reference the following two should be used: An object free spot in the preparation and a leaf tissue free from chlorophyll. The first attempt used the white part of a variegated leaf as a reference (Fig. 1). The resulting extinction curve shows maximal absorption of light between 669.2 and 671.4 nm. Above 700 nm, where no chlorophyll absorption occurs, a relatively high extinction value is still observed. This means that the chlorophyll-free structures in the green parts of a leaf show a higher absorption in comparison with the white parts. It was found that the white areas are in general 18.5% thinner than the green ones. If leaf pieces were used for reference which were previously bleached by boiling ethanol, zero extinction is measured at 750nm (Fig. 1). As the absorption of both, the green living leaf and the bleached tissue is equal at 750nm, at this wavelength the extinction of a green leaf with an adjacent empty area of the slide for reference values can serve as a standard of comparison. To get the reference values for other wavelengths the transmitted light intensity through a bleached tissue was measured at different wavelengths setting the amount of transmitted light intensity at 750 nm as 1. The decline of absorption from 550 nm to 750 nm is due to the decrease of light scattering with higher wavelength (Goldstein, 1970). As a result of several series of measurements with bleached tissues of different leaves it was proved that the relations (k~) between the transmitted light at
16
669,2 - 671,z~nm 1,5
,.'
:,
oJ
1,0 ,,.,
, .,.'''
•
I
,,.'"
I
: =:::-: :- : : 2 _
.-'"
..
'
~
•
...... :.---..
0,5
550
600
650
700 wQvelenght[nm]
%0
Fig. 1. Extinction of green Tradescantia leaves measured with a microscopic spectrophotometer: - - with artificially bleached leaf tissue for reference, - - with a white part of variegated leaf for reference, . .... with an object free area for reference, - - - , extinction of an artificially bleached leaf tissue with an object free area for reference.
750 nm and those at other wavelengths remain constant. These relations also take in consideration light scattering and differences in the spectral sensitivity o f the whole measuring device, mainly represented by the spectrum o f the measuring light and the photomultiplier.
I~(,~)
k~ -
•,(750)
-
const.
The wavelength specific factor k~ is used to calculate absorption o f leafstructure at a selected wavelength using the absorption value at 750 n m only. Therefore a bleached leaf tissue is not needed any longer to d e t e r m i n e I0 at any wavelength, 10(2)
=
11(750) " k ~ .
Selection of the measuring wavelengths In c h l o r o p h y l l estimation the extinctions are determined at the wavelengths o f m a x i m a l absorption for c h l o r o p h y l l a a n d b. The wavelengths o f these m a x i m a v a r y a c c o r d i n g to the solvent. T h e wavelength for m a x i m u m extinction o f c h l o r o p h y l l a in the living green
17 leaf tissue is determined at 670nm (see French et al., 1972). The respective wavelength for chlorophyll b is marked by a hardly detectable shoulder in the absorption curve. As a chlorophyll a solution in 15% bovine serum albumine shows the extinction maximum at 670 nm, clorophyll b was dissolved as well in BSA. The maximum extinction was found at 652 nm (see Demeter et al., 1976; Tombacz et a l., 1984). The position of the wavelength with half maximal extinction was found 15 nm above the maximum for chlorophyll a.
The two-wavelength method to correct cytophotometric errors in measurement
The errors involved in the microspectrophotometrical method are light scatter, distributional error and errors due to the spectrophotometer. By comparison of the methods of duplicated estimates, two-wavelength photometry minimises these errors (Patai, 1952; Ornstein, 1957; Garcia, 1962 a, b). 1-
7 =
1 -
1 -~ ~Fm -
2ThJ 1OgTm _
Tm- Th Tm -
T~
1 + Tm-2T h = corrected extinction value; Tm = transmittance at the wavelength with the maximal absorption (670 nm); Th = transmittance at the wavelength with half maximal absorption (685 nm and 652 nm). In theory the two-wavelength method is suitable only for solutions containing one pigment (Patzelt, 1969). However, it has been found that the corrected extinction value can be calibrated against data gained from comparable leaf material measured in conventional spectrophotometer. In this procedure the area of the photometric field is not needed for calculations. Nevertheless the photometric field should be an area with homogenous structure. The size and shape of the measuring diaphragm should be selected according to the tissue under study and should not be changed during a series of measurements. The transmittance was determined at 670 nm, 685 nm and 652 nm. The transmittances at 652nm and 685nm were used both in the calculations as Th. Therefore two corrected extinction values were recieved. The mean value of these two was used to find the corresponding chlorophyll concentrations on the calibration line (Fig. 2). The aim of this procedure was to take sufficient account of the chlorophyll b content.
The construction o f a calibration line
As stated previously the data calculated from measurements with the MPV were compared with those gained with a conventional spectrophotometer. For this
18
0,05 ff E
0,04
I *) 0,03
0,02
0,01
I
O,S
t,0
i
I
i
1,5 CORRECTED EXT. VALUES
Fig. 2. Calibration line to obtain absolute chlorophyll concentrations using values derived from living leaf in the microscopic spectralphotometer.
a
purpose about 100 measurements were taken with the MPV in a single sample. After this the total leaf area of the sample was determined and the chlorophyll was quantitatively extracted. To get different chlorophyll content in the Tradescantia leaves used, bleaching was induced by high light intensity and by the application of the herbicide atrazine. The resulting calibration line follows the equation y
=
0.0276x - 0.0006.
The correlation coefficient reaches r2 = 0.997.
19
Fig. 3. Arrangement of the measuring profiles on the leaf blade: L = longitudinal profile, C = cross profile, Dr, D L = diagonal profiles (right and left side).
To test the reliability of the equation found, leaves from several other plants were examined (Zea mays, Abutilon sp., Acalypha sp., Phaseolus vulgaris). The data received are in good agreement with the calibration line (Fig. 2). Even from leaves with a high or low ratio of chlorophyll a to b the data gained show a good correlation.
Results
The chlorophyll distribution pattern of bean leaves Plants in a complete nutrient solution (controls)
The unaided eye is not able to detect differences in the chlorophyll content of various areas of the leaf lamina of first and second trifoliate leaves. Using the MPV the highest chlorophyll concentrations (0.031 mg. cm 2) were determined in the apical part of the lamina. The leaf tip and leaf base showed values which were 5-10% and 10-15% lower, respectively. Along the diagonal profiles the chlorophyll concentration in the thickened tissue near the veins was 20-30% higher in comparison with the areoles (Fig. 4).
Plants cultivated in the dark
After 17 days under favourable conditions in the green house the plants were transferred to the dark for one week. In comparison with the controls this
20 treatment causes on average a 20% reduction in the chlorophyll concentration throughout the lamina. In single leaves the distribution pattern of the chlorophyll content varied. Some leaves showed no change in concentration at the leaf tip, others underwent a 5% higher deficit in the tissue near the veins than in the areoles.
Plants grown in an iron deficient nutrient solution
After 14 to 21 days characteristic iron deficiency symptoms will develop in plants on iron deficient media (Machold and Stephan, 1969). The second trifoliate leaves develop bleached areoles with green tissue accompanying the veins (Terry and Low, 1982). In comparison with the controls the chlorophyll concentration is generally 60% lower along the longitudinal profil. An exception is, that chlorophyll content at the leaf tip is only 30% lower. Along the diagonal profile the chlorophyll reduction increases from the central vein to the leaf margin by 20%. In the tissue along the veins the chlorophyll loss is less marked. This difference in concentration between the areoles and the tissue near the venation is three times greater as observed in the controls (Fig. 5).
rlv 120
2
i}
_ /.-/"
1O0
De
/" L °"
Dr
Fig. 4. Chlorophyll concentration in mg. cm -2 leaf area, first trifoliate leaves: . . . . . . L, nv = tissue near the veins Dr, D1, a = areoles - - - C (mid vein) 100% = 0.0293 rag. cm -2 and equals the mean of all values measured along L.
100
nv B0
60
40
n G ii
L
,I
a
/
li
De
Dr
Fig. 5. Iron deficit: Chlorophyll concentration in percent of the controls (see Fig. 4).
21
Plants treated with the herbicide Atrazine Atrazine (2-chloro-4-ethylamine-6-isopropyl-amino-s-triazine) is absorbed by leaves and roots. A concentration of 10-Smol. 1 ~ in the apoplast solution causes severe bleaching dependent on temperature and irradiation (BolharNordenkampf, 1975).
Application of Atrazine on the leaf lamina A solution containing 500 ppm atrazine (Gesaprim, Ciba-Geigy) was applied to one half of the leaf. Two days later the different profiles of each half were measured. The treated half develops an all-over chlorophyll deficit from 5-25 % with the highest losses in the areoles. The leaf tip is not influenced. The bleaching effect is restricted exclusively to the treated half of the lamina.
Application of Atrazine via the transpiration stream The plants were grown for 5 days in a complete nutrient culture solution containing 100 ppm Atrazine. After one day a chlorophyll loss of 5% became detectable at the leaf tip. Subtoxic atrazine concentrations in other parts of the lamina cause an increase in the chlorophyll content of 20% (see Krakkai, 1973). Chlorophyll concentration in the areoles does not become lower than in the controls before 5 days. In contrast the chlorphyll content of the tissue near the veins drops continuously from the second day and reaches values 10 to 20% below those of the areoles (Fig. 6).
Discussion
The enhanced chlorophyll concentration in the tissue near the vein is determined by the leaf anatomy. Along the veins the spongy parenchyma is constantly 30% thicker because of two additional cell layers. Although the minor veins near the leaf margin are considerably thinner than the main veins, the chlorophyll absorption in the accompanying parenchymatous tissue varies little. The lower chlorophyll concentration on the leaf tip is determined by a thinner
120t
--] i
a
1001 De
nv Ii
L'
--L. " --tJ G
',', Dr
Fig. 6. Complete nutrient solution containing 1000ppm Atrazine 2 days after adding herbicide (see
22 c h l o r e n c h y m a , at the leaf base the same p h e n o m e n is the result o f a c h l o r o p h y l l loss caused b y age. I r o n deficit caused a c h a n g e in the p a t t e r n o f c h l o r o p h y l l content, which d e p e n d s on the s u p p l y a n d d i s t r i b u t i o n o f the i r o n still available in the xylem sap. In general the tissue a l o n g the veins, the leaf base a n d tip are supplied better b y the xylem stream a n d therefore in these regions o f the leaf b l a d e the chlorosis is less. In the case o f an atrazine s u p p l y via the nutrient solution the herbicide is d i s t r i b u t e d in the same w a y as the iron ions. This fact leads to r a p i d bleaching on the c h l o r e n c h y m a a d j a c e n t to the veins. The c o n t r a r y effect is observed, when atrazine is a p p l i e d to the surface o f the leaf lamina. In leaf regions with a better w a t e r s u p p l y f r o m the xylem s t r e a m the diffusing herbicide is diluted a n d the bleaching is less. In c o n t r a s t the diffusive stress by placing the p l a n t s in the d a r k causes an overall bleaching with no c o r r e l a t i o n to the leaf a n a t o m y . These results show t h a t the d e v e l o p e d technique is suitable to m e a s u r e small differences in c h l o r o p h y l l d i s t r i b u t i o n on a leaf blade.
References Bolhar-Nordenkampf HR (1975) Die Ver/inderungen des Chlorophyllgehaltes in ontogenetisch verschiedenen Bl~ittern von Phaseolus vulgaris var. nanus L. nach Behandlung mit Atrazin. Biochem Physiol. Pflanzen (BPP) Bd 167:41-64 Demeter S., Sagromsky H., Faludi-Daniel A., (1976): Orientation of chlorophyll b in thylakoids of barley chloroplasts. Photosynthetica 10(2):193-197 French C.S., Brown J.S. and Lawrence M.C., (1972) Four universal forms of chlorophyll a. Plant Physiology 49:421-429 Garcia A.M., (1962) Studies in Leucocytes and related cells of mammals. I. On microspectrophotometric errors and statistical models. Histochemie 3:170-177 Garcia A.M., (1962) II. On the Feulgen reaction and Two-Wavelength microspectrophotometry. Histochemie 3:178 154 Hardacre A.K. Nicholson H.F. and Boyce M.L.P., (1984) A portable photometer for the measurement of chlorophyll in intact leaves. New Zealand Journal of Experimental Agriculture 12:357-362 Inskeep W.P. and Bloom P.R., (1985) Extinction coefficients of chlorophyll a and b in N, Ndimethylformamide and 80% acetone. Plant Physiology 77:483-485 Krakkai I. (I 973) Herbicide derived from chlortriazine as permanent stimulators of maize. Proceedings of the Research Institute of Pomology, Skierniewice, Poland Nr.3, 352-357 Machold O. and Stephan U.W., (1969) The function of iron in porphyrin and chlorophyll biosinthesis. Phytochemistry 8:2189-2192 Medina E. and Lieth H., (1964) Die Beziehungen zwischen Chlorophyllgehalt, assimilierender Flfiche und Trockensubstanzproduktion in einigen Pflanzengemeinschaften. Beitr. Biol. Pflanzen 40:451-494 Nagel W. (1940) Ober die Blattfarbstoffe des Tabak. Bot. Archiv 40:1-57 Ornstein L., (1952) The distributional error in microspectrophotometry. Lab Invest 1:250-265 Patau K., (1952) Absorption microphotometry of irregular shaped objects. Chromosoma 5, Berl. 341-362 Patzelt W.J., (1969) Absorptionsmessungen: Grundlagen und Anwendungen. Mitt.a.Labor Anw.Mikro, Mikrophotometrie, 32 a
23 Terry N and Low G (1982) Leaf chlorophyll content and its relation to the intercellular localization of iron. J Plant Nutrition 5:301-310 Tombacz E., Varonyi Z. and Szalay L., (1983) Artificial chlorophyll-protein complexes. In: Sybesma C (ed) Advances in Photosynthesis Research, Vol. 2. The Hague: Martinus Nijhoff/Dr W. Junk Publishers Ziegler R and Egle K., (1965) Zur quantitativen Analyse der Chloroplastenpigmente. I. Kritische Oberprfifung der spektralphotometrischen Chlorophyll-Bestimmung. Betr Biol Pflanzen 41:11-37 Ziegler R and Egle K. (1965b) II Verfinderungen im Chlorophyllspiegel bei ausdifferenzierten Laubbl/ittern im Laufe eines Tages. Beitr. Biol. Pflanzen 41:39-63
