Planta (Berl.)130, 33-37 (1976)
P I ~ I ] _ ~ 9 by Springer-Verlag 1976
Carbon Dioxide Assimilation in Some Aerial Plant Organs and Tissues* C.M. Willmer and W.R. Johnston Department of Biology, University of Stirling, Stirling FK9 4LA, U.K.
Summary. C O 2 fixation characteristics of a number of mature (but not senescing) tissues and organs (the outer layers of green pod and the seed testa of Vicia faba L. ; the outer layers of green pod and seeds of Trigonellafoenum-graecum L. ; the outer layers of the green fruit of Lycopersicon esculentum Mill.) were studied and compared with their respective C3 leaf characteristics. On a chlorophyll basis phosphoenolpyruvate carboxylase, malic enzyme (NADP) and malate dehydrogenase (NAD and NADP) acitivites were much higher in the non-leaf tissues (except for V. faba seed testa) than the leaf tissues. Generally, on a protein basis the differences were less significant. All tissues possessed ribulose-1.5-diphosphate carboxylase activity though there was great variation in activities both on a protein and chlorophyll basis. Protein: chlorophyll ratios varied greatly from tissue to tissue being lowest in the leaf tissue (11.5-14.0) and highest in V. faba seed testa (805.5). Chlorophyll a :b ratios were all between 2 and 3. 14CO2 uptake in the dark by L. esculentum fruit slices was about 1/3 that in the light and the major, initially labelled product was malate both in the light and dark. Neither typical C4-photosynthesis or crassulacean acid metabolism were exhibited by the non-leaf tissues and it was considered that the increased levels of certain enzyme activities were present to refix and recycle respired CO2.
all green organs or tissues on the same plant may not have the same CO/ fixation characteristics. For example, green parenchyma tissues excised from sugar cane (a C4 species) stems fixes and metabolises CO2 according to the C3 system (Kortschak and Nickell, 1970). In the C4 plant, Amaranthus retroflexus L., although malate and aspartate were the major compounds produced during a ten second exposure to 14CO2, labelled aspartate predominated in leaves while labelled malate was the major compound in stems, cotyledons and pale green callous tissue from stem pith (Usada et al., 1971). Furthermore, the primary photosynthetic product in leaves of Portulaca oleracea (a C4 species) differ considerably according to leaf age. Senescing leaves, for example, channel a substantial amount of t4c label into primary products typical of C a plants upon short-term exposure to CO2 (Kennedy and Laetsch, 1973). The CO2 fixation characteristics of green fruit and vegetables have not been studied to any great extent but there are a few references indicating that such organs may have a different COz fixation pathway from the leaf tissue. For example, high malic enzyme and pyruvate carboxylase activities were found in apples (pulp and peel), particularly at the 'climactic' period (Hulme et al., 1963). In this paper we have examined and compared the CO2 fixation characteristics of leaf tissue and tissue taken from green fruiting organs of a number of C3 species and the significance of any differences that occur are discussed.
Introduction
Higher plants can be classified into three broad groups, namely C~, C3 and CAM types, on the basis of their leaf photosynthetic characteristics. However, Abbreviations: PEP=phosphoenolpyruvate; RuDP=ribulose - 1 , 5 - , diphosphate; MDH=malate dehydrogenase; C A M = Crassulacean acid metabolism; OAA = oxaloacetic acid *
Materials and Methods The following tissues were taken from plants grown in a greenhouse: 1) Vicia faba L. (broad bean) leaf tissue from the youngest, fully expanded leaves; 2) the outer layer from expanding green pods of broad bean consisting of exocarp and the outer epidermis; 3) broad bean seed testa from developing green seeds; 4) Trigonella
34
C.M. Willmer and W.R. Johnston : Assimilation in Some Aerial Plant Organs
Table 1. Enzyme activities (micromoles substrate per mg chlorophyll or protein per hour), chlorophyll a/b and protein/chlorophyll ratios in various plant tissues. Each figure represents the mean of at least 3 determinations with its associated standard error of the mean. (ND = non-detectable) Species and tissue
PEP carboxylase
RuDP carboxylase
Malic enzyme (NADP)
MDH (NAD)
MDH (NADP)
Chl.
Prt
Chl.
Prt.
Chl.
Prt.
Chl.
Prt.
Chl.
Prt.
Prt/Chl. Chl a/b
Vicia leaf
52 +3
4.8 +1.60
368 _+69
32.10 -+ 10.10
5 +2
0.6 +0.4
2,196 _+1,018
121 _+46
18 _+8
1.0 +0.9
13.0 +2.0
2.7 -+0.1
Vicia pod (epidermis
679 _+133
9.8 _+2.80
455 6.10 _+48 _+0.60
119 _+66
1.3 _+5.0
16,400 _+2,640
196 _+37
160 2.0 _ + 1 2 0 -+7.0
91.1 _+7.0
2.6 _+0.4
Vicia seed testa
178 -+37
0.2 +0.02
140 -+71
ND
ND
18,316 _+3,637
30 _+15
ND
ND
805.5 -+94.8
2.0 _+0.3
Trigonella leaf
29 -+11
1.2 -+0.70
378 24.30 _+51 -+7.30
20 +3
1.4 _+3.0
2,407 _+320
134 _+20
60 _+11
4.4 _+0.9
14.0 _+1.9
2.6 _+0.2
Trigonella pod (epidermis + exocarp)
344 _+74
8.8 _+1.20
440 10.70 _+66 _+1.30
268 _+49
7.0 _+1.4
15,237 _+4,444
387 _+97
138 _+29
4.1 _+1.1
41.7 _+5,3
2.9 _+0.2
Trigonella seed
640 _+171
5.9 _+2.10
167 0.88 _+15 _+0.10
290 _+13
1.5 _+0.4
35,708 _+6,158
206 _+15
614 _+26
3.0 _+0.2
127.3 +39.5
2.6 _+0.1
Lycopersicon leaf
12 _+1
0.9 _+0.10
187 +34
14.50 _+3.30
7 _+1
0.9 _+0.5
1,590 _+572
176 _+53
42 +3
3.5 -+0.3
11.5 +0.9
2.5 -+0.1
Lycopersicon fruit
3,147 27.8 -+1,052 -+9.00
302 -+20
2.00 _+0.30
687 _+152
3.3 _+0.8
93,934 _+10,180
348 _+19
1,278 5.0 _ + 3 7 8 +0.8
194.1 -+19.2
2.4 _+0.3
+ exocarp.)
(epidermis+Mesocarp).
0.16 _+0.10
foenum-graecum L. (fenugreek) leaf tissue from the youngest fully expanded leaves; 5) the outer layer form expanding green pods of fenugreek consisting of the epidermal layer and exocarp; 6) developing fenugreek seeds consisting largely of two green cotyledons and the testa; 7) Lyeopersicon eseulentum Mill. (tomato) leaf tissue from the youngest fully expanded leaves; 8) the outer layer of green tomato fruit consisting of the outer epidermal layer and mesocarp.
Preparation of Extracts for Enzyme Assays. The tissue samples were finely sliced and ground in an ice-cold mortar containing the following medium: 50 mM tris buffer, pH 8.3; 1 mM EDTA; 5 mM MgC12 and 5 mM dithiothreitol. Tissue homogenates were filtered through 25 or 30 txm nylon nets and the filtrate used for the enzyme assays. Enzyme assays were conducted using standard procedures and were identical to those reported by Willmer et al. (1973). PEP-carboxylase (EC 4.1.1.31) and RuDP carboxylase (EC 4.1.1.39) activities were assayed by measuring 14C incorporation from N a i l 14CO3 solutions. Assays for NADP malic enzyme (EC 1.1.1.40) and MDH, (EC 1.1.1.37) NAD and NADP specific, were determined spectrophotmetrically following the change in absorbance at 340 nm at room temperature. Chlorophyll determinations followed the method of Winterroans and De Mots (1965). Total soluble protein was determined as follows. One ml of extract was added to 0.5 ml of 10% trichloroacetic acid and centrifuged on a bench centrifuge for five minutes at maximum speed. The supernatant was discarded and one ml 1.0 N NaOH added to dissolve the precipitated protein. The Folin method (Lowry et al., 1951) was then conducted to estimate the protein content. Rates of CO2 uptake and the distribution of radioactivity among labelled compounds in the outer layers of tomato fruit tissue were determined in the light and dark as follows. The tissue was finely sliced with a razor blade and immersed in 2.0 ml of 0.1 mM CaSO~. After a pre-illumination or pre-dark period of
ten minutes one mCi NaH14COa (0.5 ml) was injected into the medium. The tissue was then incubated in the light or dark, being constantly agitated with a magnetic stirrer bar. At various intervals up to 120 s a sample of the tissue was extracted and immediately placed in 2.0 ml of hot glacial acetic acid-ethanol mixture to stop the reaction. The tissue was extracted in 75% ethanol. Two dimensional chromatography was performed with the extracts using standard descending paper chromotographic procedures (Bassham and Calvin, 1957). The amounts of labelled soluble and insoluble products were determined by liquid scintillation counting (Packard Tri-carb Liquid Scintillation Spectrophotometer, Model 2425). Measurements of CO2 compensation points were made on fully expanded excised leaves with their petioles cut under airfree water. Three determinations were made on separate samples for each organ or tissue. A leaf was placed into the sample chamber and left in the closed system until the compensation point was established. The sample chamber consisted of a perspex box which was immersed in a constant temperature bath at 26-27 ~ C. The chamber was connected by glass tubing to a mercury sealed pulsating pump and a Grubb Parsons infrared gas analyser (Serial number 3048). Illumination was from a mercury vapour lamp giving an intensity of about 750 geinsteins/m 2 (-~ 13,000 lux). Stomatal frequencies were determined as follows. Three epidermal or paradermal sections were made and the number of stomata counted in 10 fields of view (0.057 mm 2) in each section.
Results and Discussion Table 1 summarises the levels of the enzyme activities in the various tissues on protein and chlorophyll bases; chlorophyll a :b ratios and protein :chlorophyll ratios are also given for the various tissues. Considerable variation of certain enzyme activities in some
C.M. Willmer and W.R. Johnston: Assimilation in Some Aerial Plant Organs
tissues occurred (see associated standard errors of the means). This is probably due to differences in the developmental stage of the tissue since enzyme levels are likely to change as the tissue or organ develops. Although efforts were made to use material uniform in size and age some variation of these parameters would occur. Except for the broad bean seed testa, higher levels of PEP carboxylase, NADP, M D H and NADP malic enzyme were found in non-leaf tissues on a chlorophyll basis and, generally, on a protein basis. N A D M D H activities were much higher in the non-leaf tissues on a protein and chlorophyll basis (with the exception of the broad bean seed testa on a protein basis) than in the leaf tissues. This was particularly apparent in the tomato fruit, fenugreek seed and pod tissues. RuDP carboxylase could be detected in all of the tissues investigated including the broad bean seed testa. In broad bean seed testa RuDP carboxylase activity was significant on a chlorophyll basis though very low on a protein basis. RuDP carboxylase activity has previously been detected in the endosperm of germinating castor beans (Benedict, 1973) apparently localised in the 'proplastids' (Osmond et al., 1975). The latter workers concluded that its presence was due to incomplete suppression of the enzyme system normally restricted to the chloroplasts of the photosynthetic tissue; its purpose in the endosperm, however, was not clear. Protein: chlorophyll ratios varied greatly between the various tissues being between 11.5-14.0 for the leaf tissues and as high as 805.5 for broad bean seed testa. Because of this wide variation the enzyme activities were expressed on protein and chlorophyll bases to afford some measure of comparison between tissues. Many of the enzyme activities, however, were at much higher levels in the non-leaf tissues than in the leaf tissues whether expressed on a chlorophyll or a protein basis. Certain of the enzymes involved in CO/ fixation and metabolism, namely PEP carboxylase, NADP malic enzyme and NADP MDH, were consistently higher (on a protein and chlorophyll basis) in the non-leaf tissues than in the leaf tissues. In tomato fruit tissue levels of these enzymes were particularly high. Such high levels of PEP carboylase, NADP malic enzyme and NADP M D H found in some of the non-leaf tissues are more typical of certain C4 and CAM leaf tissues. The leaf tissues possessed very low levels of activity of PEP carboxylase, NADP malic enzyme and NADP M D H and considerable activity of RuDP carboxylase typical of C3-plants. Accordingly, the plants ur{der investigation all gave leaf COz compensation points typical of C3 species
35
F
,oF / .ok/ 0 w ~
0
20
/ I
I
I
I
40
60
80
zoo
I
120
Time (Secs)
Fig. 1. 14CO2 fixation kinetics of the outer layers of green tomato fruit tissue in light (o) and darkness (e)
being, for tomato leaf, fenugreek leaf and broad bean leaf, 42, 39 and 59 lal/1 CO2, respectively. Chlorophyll a:b ratios for all the tissues were between 2 and 3, typical of C3 leaf (Black and Mayne, 1970) and CAM leaf (Rouhani, 1972) tissues. Further investigations into the pathway of CO2 fixation and metabolism were made with tomato fruit slices. 14CO2 uptake studies in the light and dark were made and the distribution of labelled compounds observed. Figure 1 shows the relatively high uptake rate of 14C02in the dark (about 1/3 that of light uptake) atypical of C-3 photosynthesis. Figure 2 shows the changes in the distribution of radioactivity among 14C-labelled compounds during fixation in the light (Fig. 2A) and dark (Fig. 2B). The major compound labelled in the light and dark was malate. In the light the level of labelled malate dropped with time with concommitant increase of label in sugar mono- and diphosphates and glutamine and alanine. After 10s exposure to ~4CO2 about 20% of the label was found in PGA which also tended to drop with time. In the dark malate, citrate andaspartate were the only labelled compounds detectable and the amount of label in each changed little with time. These data suggest that CO2 is fixed by PEP carboxylase into OAA which in turn is converted to malic acid via NADP MDH. Whether the phosphorylated compounds formed in the light are derived from malate (via malic enzyme activity) or whether they are formed from a parallel and direct (without CO2
36
C.M. Willmer and W.R. Johnston: Assimilation in Some Aerial Plant Organs I00 80 60
"
~
e
MALATE
40
OTHERS
o
2O la. O
~-
A
/
e
~
I,
0
-
I
'
~ I
SUGAR P
I
I
I
I
B
3 80
~e
MALATI=e
60 40-
CITRATE
20-
9~
.
\ ASP
O
0
10
20
30
40
Time
(Sees)
50
60
70
Fig. 2A and B. Changes in the distribution of radioactivity among 14C-labelled compounds in the outer layers of green tomato fruit tissue fixing ~4CO2 in the light, (A), and dark, (B)
Table 2. CO2 compensation points and stomatal frequencies in
the epidermis of various plant organs. The CO2 compensation points represent the means of 3 determinations and the standard errors of the means are given where possible. Thirty counts (10 per epidermal section) in a viewing area of 0.057mm 2 were made to obtain the stomatal frequency Species and tissue
CO2 compensation point (pl/1)
Stomatal frequency (stomata/mm2) upper surface
Vicia leaf Vicia pod Vicia seed Trigonella leaf TrigoneIla pod Trigonella seed Lycopersicon leaf Lycopersicon fruit
59+ 1 > 240 > 240 39+2 > 240 > 240 42_+ 1 > 240
lower surface
38+3
70+3 19+_2 0
245+15
225+_11 61 + 4 0
34 + 5
203 + 9 0
being channelled through C4 acids) carboxylation via RuDP carboxylase is not known. Such a labelling pattern is typical of C4 photosynthesis or of CAM at certain periods of CO2 assimilation. The chlorophyll a:b ratios, the obvious lack o f ' Kranz' type anatomy and the inability to maintain a CO2 compensation point (Table 2) in the non-leaf tissues, however, suggest the absence of C4-photosynthesis. In order to determine if the labelling pattern
in tomato fruit represented a type of CAM, both excised tomato fruits and plants with tomato fruits attached were placed in the dark or the light for 21 hours and the titratable acids and cell sap pH of the outer layers of the fruits determined. No significant fluctuations in acidity were observed in tissue from detached tomato fruits or from fruits attached to plants. The pH of the cell sap was 5.5 and 5.4 at the end of the light and dark periods, respectively, for detached fruits and remained at 4:7 when fruits were attached to plants. Fluctuations in titratable acids did not occur either indicating the absence of CAM. Leaves from plants of KalanchoO daigrernontiana exposed to the same treatments showed large fluctuations in pH of the cell sap (pH 3.6 and 4.6 at the end of the dark and light periods, respectively) and titratable acids. Thus, these aerial, non-leaf tissues and organs fix and metabolise CO2 in a manner different to their leaf tissues (which exhibit C3-photosynthesis ) and resemble C4 or CAM tissues regarding their levels of certain enzymes and l~C-labelling patterns but differ in certain anatomical and physiological characteristics. The respiratory rates of the non-leaf tissues, as gauged by NAD M D H activities, were, generally, much greater than in the leaf tissues. Thus, with this high metabolic activity there will be an associated high production of CO2. This is reflected in the observation that there is a constant loss of COg from the surface of the non-leaf tissues (which possess few or no stomata in their epidermes) and the inability of the tissues to reach a CO2 compensation point (Table 2). In tomato fruit, for example, the epidermis possesses a thick cuticle and thick-walled epidermal cells with no stomata although there was continual loss of COg from the fruit indicating the steepness of the COz concentration gradient between the inside and outside of the fruit. It is suggested that the high levels of PEP carboxylase in tomato fruit tissue and the other non-leaf tissues is present to refix the respired CO2 thereby minimising the losses of CO2 and making the organs more efficient. There is some evidence also that the pod tissues of Phaseolus vulgaris (Crookston et al., 1974) and the reproductive structures of Glycine max (Quebedeux and Chollet, 1975) are involved in reassimilating respired CO2. It is evident that the organic acids do not accumulate to any extent, at least in tomato fruit tissue, since there was no diurnal fluctuation of cell sap pH or the levels of titrable acids. This is surprising in view of the relatively high CO2 fixation rates in the dark (Fig. 1). Possibly the" tissue has a high buffering capacity or, in absolute terms, CO2 fixation was low
C.M. Willmer and W.R. Johnston: Assimilation in Some Aerial Plant Organs
and little malic acid was produced (even though it was the maj or compound labelled). Another obscurity was the purpose of the relatively high malic enzyme activity in the non-leaf tissues since, as a decarboxylase, CO2 would be produced from malate adding to that appearing from respiratory processes. Such problems need to be resolved and further investigations made before it can be concluded that the enzymes and CO2 fixation pathway studied in this paper are primarily concerned in refixing respired CO2 or are part of a major pathway of carbohydrate metabolism in the developing non-leaf tissues. The authors wish to thank Dr. P. Dittrich for his valuable suggestions and advice and Mr. A. Hodge for his competent technical assistance.
References Bassham, J.A., Calvin, M. : The path of carbon in photosynthesis. Englewood Cliffs, N.J. : Prentice Hall, Inc. (1957) Benedict, C.R. : The presence of ributose-l, 5-diphosphate carboxylase in the nonphotosynthetic endosperm of germinating castor beans. Plant Physiol. 36, 755-759 (1973) Black, C.C., Jr., Mayne, B.C. : PToo activity and chlorophyll content of plants with different photosynthetic carbon dioxide fixation cycles. Plant Physiol. 45, 738-741 (1970) Crookston, R.K., O'Toole, J., Ozbun, J.L.: Characterization of the bean pod as a photosynthetic organ. Crop Sci. 14, 708-712 (1974)
37
Hulme, A.C., Jones, J.D., Wooltorton, L.S.C.: The respiration climacteric in apple fruits. Proc. Roy. Soc. B158, 514-535 (1963) Kennedy, R.A., Laetsch, W.M.: Relationship between leaf development and primary photosynthetic products in the C 4 plant Portulaca oleracea L. Planta 115, 113-124 (1973) Kortschak, H.P., Nickell, L.G. : Calvin-type carbon dioxide fixation in sugarcane stalk parenchyma tissue. Plant Physiol. 45, 515-516 (1970) Lowry, O.H., Farr, A.L, Rosebrough, N.J., Randall, R.J. : Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265~75 (1951) Osmond, C.B., Akazawa, T., Beevers, H. : Localization and properties ofribulose diphosphate carboxylase from castor bean endosperm. Plant Physiol. 55, 226-238 (1975) Quebedeaux, B., Chollet, R. : Growth and development of soybean (Glycine max L. Merr.) pods. CO 2 exchange and enzyme studies. Plant Physiol. 55, 745-748 (1975) Rouhani, I. : Pathways of carbon metabolism in spongy mesophyll cells, isolated from Sedum telephium leaves, and their relationship to Crassulacean Acid Metabolism. Ph.D. Thesis, Univ. Georgia (1972) Willmer, C.M., Pallas, J.E., Jr., Black, C.C., Jr.: Carbon dioxide metabolism in leaf epidermal tissue. Plant Physiol. 52, 448452 (1973) Wintermans, J.F.G.M., DeMots, A. : Spectrophotometric characteristics of chlorophyll and their pheophytins in ethanol. Biochim. Biophys. Acta (Amst.) 109, 448453 (i965) Usada, H., Kanai, R., Takeuchi, M.: Comparison of carbon dioxide fixation and the fine structure in various assimilatory tissues of Amaranthus retroflexus L. Plant Cell Physiol. 12, 917 930 (1971)
Received 14 November; accepted 9 December 1975
P I ~ I ] _ ~ 9 by Springer-Verlag 1976
Carbon Dioxide Assimilation in Some Aerial Plant Organs and Tissues* C.M. Willmer and W.R. Johnston Department of Biology, University of Stirling, Stirling FK9 4LA, U.K.
Summary. C O 2 fixation characteristics of a number of mature (but not senescing) tissues and organs (the outer layers of green pod and the seed testa of Vicia faba L. ; the outer layers of green pod and seeds of Trigonellafoenum-graecum L. ; the outer layers of the green fruit of Lycopersicon esculentum Mill.) were studied and compared with their respective C3 leaf characteristics. On a chlorophyll basis phosphoenolpyruvate carboxylase, malic enzyme (NADP) and malate dehydrogenase (NAD and NADP) acitivites were much higher in the non-leaf tissues (except for V. faba seed testa) than the leaf tissues. Generally, on a protein basis the differences were less significant. All tissues possessed ribulose-1.5-diphosphate carboxylase activity though there was great variation in activities both on a protein and chlorophyll basis. Protein: chlorophyll ratios varied greatly from tissue to tissue being lowest in the leaf tissue (11.5-14.0) and highest in V. faba seed testa (805.5). Chlorophyll a :b ratios were all between 2 and 3. 14CO2 uptake in the dark by L. esculentum fruit slices was about 1/3 that in the light and the major, initially labelled product was malate both in the light and dark. Neither typical C4-photosynthesis or crassulacean acid metabolism were exhibited by the non-leaf tissues and it was considered that the increased levels of certain enzyme activities were present to refix and recycle respired CO2.
all green organs or tissues on the same plant may not have the same CO/ fixation characteristics. For example, green parenchyma tissues excised from sugar cane (a C4 species) stems fixes and metabolises CO2 according to the C3 system (Kortschak and Nickell, 1970). In the C4 plant, Amaranthus retroflexus L., although malate and aspartate were the major compounds produced during a ten second exposure to 14CO2, labelled aspartate predominated in leaves while labelled malate was the major compound in stems, cotyledons and pale green callous tissue from stem pith (Usada et al., 1971). Furthermore, the primary photosynthetic product in leaves of Portulaca oleracea (a C4 species) differ considerably according to leaf age. Senescing leaves, for example, channel a substantial amount of t4c label into primary products typical of C a plants upon short-term exposure to CO2 (Kennedy and Laetsch, 1973). The CO2 fixation characteristics of green fruit and vegetables have not been studied to any great extent but there are a few references indicating that such organs may have a different COz fixation pathway from the leaf tissue. For example, high malic enzyme and pyruvate carboxylase activities were found in apples (pulp and peel), particularly at the 'climactic' period (Hulme et al., 1963). In this paper we have examined and compared the CO2 fixation characteristics of leaf tissue and tissue taken from green fruiting organs of a number of C3 species and the significance of any differences that occur are discussed.
Introduction
Higher plants can be classified into three broad groups, namely C~, C3 and CAM types, on the basis of their leaf photosynthetic characteristics. However, Abbreviations: PEP=phosphoenolpyruvate; RuDP=ribulose - 1 , 5 - , diphosphate; MDH=malate dehydrogenase; C A M = Crassulacean acid metabolism; OAA = oxaloacetic acid *
Materials and Methods The following tissues were taken from plants grown in a greenhouse: 1) Vicia faba L. (broad bean) leaf tissue from the youngest, fully expanded leaves; 2) the outer layer from expanding green pods of broad bean consisting of exocarp and the outer epidermis; 3) broad bean seed testa from developing green seeds; 4) Trigonella
34
C.M. Willmer and W.R. Johnston : Assimilation in Some Aerial Plant Organs
Table 1. Enzyme activities (micromoles substrate per mg chlorophyll or protein per hour), chlorophyll a/b and protein/chlorophyll ratios in various plant tissues. Each figure represents the mean of at least 3 determinations with its associated standard error of the mean. (ND = non-detectable) Species and tissue
PEP carboxylase
RuDP carboxylase
Malic enzyme (NADP)
MDH (NAD)
MDH (NADP)
Chl.
Prt
Chl.
Prt.
Chl.
Prt.
Chl.
Prt.
Chl.
Prt.
Prt/Chl. Chl a/b
Vicia leaf
52 +3
4.8 +1.60
368 _+69
32.10 -+ 10.10
5 +2
0.6 +0.4
2,196 _+1,018
121 _+46
18 _+8
1.0 +0.9
13.0 +2.0
2.7 -+0.1
Vicia pod (epidermis
679 _+133
9.8 _+2.80
455 6.10 _+48 _+0.60
119 _+66
1.3 _+5.0
16,400 _+2,640
196 _+37
160 2.0 _ + 1 2 0 -+7.0
91.1 _+7.0
2.6 _+0.4
Vicia seed testa
178 -+37
0.2 +0.02
140 -+71
ND
ND
18,316 _+3,637
30 _+15
ND
ND
805.5 -+94.8
2.0 _+0.3
Trigonella leaf
29 -+11
1.2 -+0.70
378 24.30 _+51 -+7.30
20 +3
1.4 _+3.0
2,407 _+320
134 _+20
60 _+11
4.4 _+0.9
14.0 _+1.9
2.6 _+0.2
Trigonella pod (epidermis + exocarp)
344 _+74
8.8 _+1.20
440 10.70 _+66 _+1.30
268 _+49
7.0 _+1.4
15,237 _+4,444
387 _+97
138 _+29
4.1 _+1.1
41.7 _+5,3
2.9 _+0.2
Trigonella seed
640 _+171
5.9 _+2.10
167 0.88 _+15 _+0.10
290 _+13
1.5 _+0.4
35,708 _+6,158
206 _+15
614 _+26
3.0 _+0.2
127.3 +39.5
2.6 _+0.1
Lycopersicon leaf
12 _+1
0.9 _+0.10
187 +34
14.50 _+3.30
7 _+1
0.9 _+0.5
1,590 _+572
176 _+53
42 +3
3.5 -+0.3
11.5 +0.9
2.5 -+0.1
Lycopersicon fruit
3,147 27.8 -+1,052 -+9.00
302 -+20
2.00 _+0.30
687 _+152
3.3 _+0.8
93,934 _+10,180
348 _+19
1,278 5.0 _ + 3 7 8 +0.8
194.1 -+19.2
2.4 _+0.3
+ exocarp.)
(epidermis+Mesocarp).
0.16 _+0.10
foenum-graecum L. (fenugreek) leaf tissue from the youngest fully expanded leaves; 5) the outer layer form expanding green pods of fenugreek consisting of the epidermal layer and exocarp; 6) developing fenugreek seeds consisting largely of two green cotyledons and the testa; 7) Lyeopersicon eseulentum Mill. (tomato) leaf tissue from the youngest fully expanded leaves; 8) the outer layer of green tomato fruit consisting of the outer epidermal layer and mesocarp.
Preparation of Extracts for Enzyme Assays. The tissue samples were finely sliced and ground in an ice-cold mortar containing the following medium: 50 mM tris buffer, pH 8.3; 1 mM EDTA; 5 mM MgC12 and 5 mM dithiothreitol. Tissue homogenates were filtered through 25 or 30 txm nylon nets and the filtrate used for the enzyme assays. Enzyme assays were conducted using standard procedures and were identical to those reported by Willmer et al. (1973). PEP-carboxylase (EC 4.1.1.31) and RuDP carboxylase (EC 4.1.1.39) activities were assayed by measuring 14C incorporation from N a i l 14CO3 solutions. Assays for NADP malic enzyme (EC 1.1.1.40) and MDH, (EC 1.1.1.37) NAD and NADP specific, were determined spectrophotmetrically following the change in absorbance at 340 nm at room temperature. Chlorophyll determinations followed the method of Winterroans and De Mots (1965). Total soluble protein was determined as follows. One ml of extract was added to 0.5 ml of 10% trichloroacetic acid and centrifuged on a bench centrifuge for five minutes at maximum speed. The supernatant was discarded and one ml 1.0 N NaOH added to dissolve the precipitated protein. The Folin method (Lowry et al., 1951) was then conducted to estimate the protein content. Rates of CO2 uptake and the distribution of radioactivity among labelled compounds in the outer layers of tomato fruit tissue were determined in the light and dark as follows. The tissue was finely sliced with a razor blade and immersed in 2.0 ml of 0.1 mM CaSO~. After a pre-illumination or pre-dark period of
ten minutes one mCi NaH14COa (0.5 ml) was injected into the medium. The tissue was then incubated in the light or dark, being constantly agitated with a magnetic stirrer bar. At various intervals up to 120 s a sample of the tissue was extracted and immediately placed in 2.0 ml of hot glacial acetic acid-ethanol mixture to stop the reaction. The tissue was extracted in 75% ethanol. Two dimensional chromatography was performed with the extracts using standard descending paper chromotographic procedures (Bassham and Calvin, 1957). The amounts of labelled soluble and insoluble products were determined by liquid scintillation counting (Packard Tri-carb Liquid Scintillation Spectrophotometer, Model 2425). Measurements of CO2 compensation points were made on fully expanded excised leaves with their petioles cut under airfree water. Three determinations were made on separate samples for each organ or tissue. A leaf was placed into the sample chamber and left in the closed system until the compensation point was established. The sample chamber consisted of a perspex box which was immersed in a constant temperature bath at 26-27 ~ C. The chamber was connected by glass tubing to a mercury sealed pulsating pump and a Grubb Parsons infrared gas analyser (Serial number 3048). Illumination was from a mercury vapour lamp giving an intensity of about 750 geinsteins/m 2 (-~ 13,000 lux). Stomatal frequencies were determined as follows. Three epidermal or paradermal sections were made and the number of stomata counted in 10 fields of view (0.057 mm 2) in each section.
Results and Discussion Table 1 summarises the levels of the enzyme activities in the various tissues on protein and chlorophyll bases; chlorophyll a :b ratios and protein :chlorophyll ratios are also given for the various tissues. Considerable variation of certain enzyme activities in some
C.M. Willmer and W.R. Johnston: Assimilation in Some Aerial Plant Organs
tissues occurred (see associated standard errors of the means). This is probably due to differences in the developmental stage of the tissue since enzyme levels are likely to change as the tissue or organ develops. Although efforts were made to use material uniform in size and age some variation of these parameters would occur. Except for the broad bean seed testa, higher levels of PEP carboxylase, NADP, M D H and NADP malic enzyme were found in non-leaf tissues on a chlorophyll basis and, generally, on a protein basis. N A D M D H activities were much higher in the non-leaf tissues on a protein and chlorophyll basis (with the exception of the broad bean seed testa on a protein basis) than in the leaf tissues. This was particularly apparent in the tomato fruit, fenugreek seed and pod tissues. RuDP carboxylase could be detected in all of the tissues investigated including the broad bean seed testa. In broad bean seed testa RuDP carboxylase activity was significant on a chlorophyll basis though very low on a protein basis. RuDP carboxylase activity has previously been detected in the endosperm of germinating castor beans (Benedict, 1973) apparently localised in the 'proplastids' (Osmond et al., 1975). The latter workers concluded that its presence was due to incomplete suppression of the enzyme system normally restricted to the chloroplasts of the photosynthetic tissue; its purpose in the endosperm, however, was not clear. Protein: chlorophyll ratios varied greatly between the various tissues being between 11.5-14.0 for the leaf tissues and as high as 805.5 for broad bean seed testa. Because of this wide variation the enzyme activities were expressed on protein and chlorophyll bases to afford some measure of comparison between tissues. Many of the enzyme activities, however, were at much higher levels in the non-leaf tissues than in the leaf tissues whether expressed on a chlorophyll or a protein basis. Certain of the enzymes involved in CO/ fixation and metabolism, namely PEP carboxylase, NADP malic enzyme and NADP MDH, were consistently higher (on a protein and chlorophyll basis) in the non-leaf tissues than in the leaf tissues. In tomato fruit tissue levels of these enzymes were particularly high. Such high levels of PEP carboylase, NADP malic enzyme and NADP M D H found in some of the non-leaf tissues are more typical of certain C4 and CAM leaf tissues. The leaf tissues possessed very low levels of activity of PEP carboxylase, NADP malic enzyme and NADP M D H and considerable activity of RuDP carboxylase typical of C3-plants. Accordingly, the plants ur{der investigation all gave leaf COz compensation points typical of C3 species
35
F
,oF / .ok/ 0 w ~
0
20
/ I
I
I
I
40
60
80
zoo
I
120
Time (Secs)
Fig. 1. 14CO2 fixation kinetics of the outer layers of green tomato fruit tissue in light (o) and darkness (e)
being, for tomato leaf, fenugreek leaf and broad bean leaf, 42, 39 and 59 lal/1 CO2, respectively. Chlorophyll a:b ratios for all the tissues were between 2 and 3, typical of C3 leaf (Black and Mayne, 1970) and CAM leaf (Rouhani, 1972) tissues. Further investigations into the pathway of CO2 fixation and metabolism were made with tomato fruit slices. 14CO2 uptake studies in the light and dark were made and the distribution of labelled compounds observed. Figure 1 shows the relatively high uptake rate of 14C02in the dark (about 1/3 that of light uptake) atypical of C-3 photosynthesis. Figure 2 shows the changes in the distribution of radioactivity among 14C-labelled compounds during fixation in the light (Fig. 2A) and dark (Fig. 2B). The major compound labelled in the light and dark was malate. In the light the level of labelled malate dropped with time with concommitant increase of label in sugar mono- and diphosphates and glutamine and alanine. After 10s exposure to ~4CO2 about 20% of the label was found in PGA which also tended to drop with time. In the dark malate, citrate andaspartate were the only labelled compounds detectable and the amount of label in each changed little with time. These data suggest that CO2 is fixed by PEP carboxylase into OAA which in turn is converted to malic acid via NADP MDH. Whether the phosphorylated compounds formed in the light are derived from malate (via malic enzyme activity) or whether they are formed from a parallel and direct (without CO2
36
C.M. Willmer and W.R. Johnston: Assimilation in Some Aerial Plant Organs I00 80 60
"
~
e
MALATE
40
OTHERS
o
2O la. O
~-
A
/
e
~
I,
0
-
I
'
~ I
SUGAR P
I
I
I
I
B
3 80
~e
MALATI=e
60 40-
CITRATE
20-
9~
.
\ ASP
O
0
10
20
30
40
Time
(Sees)
50
60
70
Fig. 2A and B. Changes in the distribution of radioactivity among 14C-labelled compounds in the outer layers of green tomato fruit tissue fixing ~4CO2 in the light, (A), and dark, (B)
Table 2. CO2 compensation points and stomatal frequencies in
the epidermis of various plant organs. The CO2 compensation points represent the means of 3 determinations and the standard errors of the means are given where possible. Thirty counts (10 per epidermal section) in a viewing area of 0.057mm 2 were made to obtain the stomatal frequency Species and tissue
CO2 compensation point (pl/1)
Stomatal frequency (stomata/mm2) upper surface
Vicia leaf Vicia pod Vicia seed Trigonella leaf TrigoneIla pod Trigonella seed Lycopersicon leaf Lycopersicon fruit
59+ 1 > 240 > 240 39+2 > 240 > 240 42_+ 1 > 240
lower surface
38+3
70+3 19+_2 0
245+15
225+_11 61 + 4 0
34 + 5
203 + 9 0
being channelled through C4 acids) carboxylation via RuDP carboxylase is not known. Such a labelling pattern is typical of C4 photosynthesis or of CAM at certain periods of CO2 assimilation. The chlorophyll a:b ratios, the obvious lack o f ' Kranz' type anatomy and the inability to maintain a CO2 compensation point (Table 2) in the non-leaf tissues, however, suggest the absence of C4-photosynthesis. In order to determine if the labelling pattern
in tomato fruit represented a type of CAM, both excised tomato fruits and plants with tomato fruits attached were placed in the dark or the light for 21 hours and the titratable acids and cell sap pH of the outer layers of the fruits determined. No significant fluctuations in acidity were observed in tissue from detached tomato fruits or from fruits attached to plants. The pH of the cell sap was 5.5 and 5.4 at the end of the light and dark periods, respectively, for detached fruits and remained at 4:7 when fruits were attached to plants. Fluctuations in titratable acids did not occur either indicating the absence of CAM. Leaves from plants of KalanchoO daigrernontiana exposed to the same treatments showed large fluctuations in pH of the cell sap (pH 3.6 and 4.6 at the end of the dark and light periods, respectively) and titratable acids. Thus, these aerial, non-leaf tissues and organs fix and metabolise CO2 in a manner different to their leaf tissues (which exhibit C3-photosynthesis ) and resemble C4 or CAM tissues regarding their levels of certain enzymes and l~C-labelling patterns but differ in certain anatomical and physiological characteristics. The respiratory rates of the non-leaf tissues, as gauged by NAD M D H activities, were, generally, much greater than in the leaf tissues. Thus, with this high metabolic activity there will be an associated high production of CO2. This is reflected in the observation that there is a constant loss of COg from the surface of the non-leaf tissues (which possess few or no stomata in their epidermes) and the inability of the tissues to reach a CO2 compensation point (Table 2). In tomato fruit, for example, the epidermis possesses a thick cuticle and thick-walled epidermal cells with no stomata although there was continual loss of COg from the fruit indicating the steepness of the COz concentration gradient between the inside and outside of the fruit. It is suggested that the high levels of PEP carboxylase in tomato fruit tissue and the other non-leaf tissues is present to refix the respired CO2 thereby minimising the losses of CO2 and making the organs more efficient. There is some evidence also that the pod tissues of Phaseolus vulgaris (Crookston et al., 1974) and the reproductive structures of Glycine max (Quebedeux and Chollet, 1975) are involved in reassimilating respired CO2. It is evident that the organic acids do not accumulate to any extent, at least in tomato fruit tissue, since there was no diurnal fluctuation of cell sap pH or the levels of titrable acids. This is surprising in view of the relatively high CO2 fixation rates in the dark (Fig. 1). Possibly the" tissue has a high buffering capacity or, in absolute terms, CO2 fixation was low
C.M. Willmer and W.R. Johnston: Assimilation in Some Aerial Plant Organs
and little malic acid was produced (even though it was the maj or compound labelled). Another obscurity was the purpose of the relatively high malic enzyme activity in the non-leaf tissues since, as a decarboxylase, CO2 would be produced from malate adding to that appearing from respiratory processes. Such problems need to be resolved and further investigations made before it can be concluded that the enzymes and CO2 fixation pathway studied in this paper are primarily concerned in refixing respired CO2 or are part of a major pathway of carbohydrate metabolism in the developing non-leaf tissues. The authors wish to thank Dr. P. Dittrich for his valuable suggestions and advice and Mr. A. Hodge for his competent technical assistance.
References Bassham, J.A., Calvin, M. : The path of carbon in photosynthesis. Englewood Cliffs, N.J. : Prentice Hall, Inc. (1957) Benedict, C.R. : The presence of ributose-l, 5-diphosphate carboxylase in the nonphotosynthetic endosperm of germinating castor beans. Plant Physiol. 36, 755-759 (1973) Black, C.C., Jr., Mayne, B.C. : PToo activity and chlorophyll content of plants with different photosynthetic carbon dioxide fixation cycles. Plant Physiol. 45, 738-741 (1970) Crookston, R.K., O'Toole, J., Ozbun, J.L.: Characterization of the bean pod as a photosynthetic organ. Crop Sci. 14, 708-712 (1974)
37
Hulme, A.C., Jones, J.D., Wooltorton, L.S.C.: The respiration climacteric in apple fruits. Proc. Roy. Soc. B158, 514-535 (1963) Kennedy, R.A., Laetsch, W.M.: Relationship between leaf development and primary photosynthetic products in the C 4 plant Portulaca oleracea L. Planta 115, 113-124 (1973) Kortschak, H.P., Nickell, L.G. : Calvin-type carbon dioxide fixation in sugarcane stalk parenchyma tissue. Plant Physiol. 45, 515-516 (1970) Lowry, O.H., Farr, A.L, Rosebrough, N.J., Randall, R.J. : Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265~75 (1951) Osmond, C.B., Akazawa, T., Beevers, H. : Localization and properties ofribulose diphosphate carboxylase from castor bean endosperm. Plant Physiol. 55, 226-238 (1975) Quebedeaux, B., Chollet, R. : Growth and development of soybean (Glycine max L. Merr.) pods. CO 2 exchange and enzyme studies. Plant Physiol. 55, 745-748 (1975) Rouhani, I. : Pathways of carbon metabolism in spongy mesophyll cells, isolated from Sedum telephium leaves, and their relationship to Crassulacean Acid Metabolism. Ph.D. Thesis, Univ. Georgia (1972) Willmer, C.M., Pallas, J.E., Jr., Black, C.C., Jr.: Carbon dioxide metabolism in leaf epidermal tissue. Plant Physiol. 52, 448452 (1973) Wintermans, J.F.G.M., DeMots, A. : Spectrophotometric characteristics of chlorophyll and their pheophytins in ethanol. Biochim. Biophys. Acta (Amst.) 109, 448453 (i965) Usada, H., Kanai, R., Takeuchi, M.: Comparison of carbon dioxide fixation and the fine structure in various assimilatory tissues of Amaranthus retroflexus L. Plant Cell Physiol. 12, 917 930 (1971)
Received 14 November; accepted 9 December 1975
