Bioluminescence produced by the luciferin-luciferase system is a very sensitive assay for ATP content in extracts of plant materials. The ATP test for seed and pollen viability and vigor is presented, along with prediction of high growth potential and productivity in new crosses and selections of breeding materials. ATP as an indicator for environmental quality, stresses, and metabolic regulation is also considered

Ching, T. M.

English

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BIOLUMINESCENCE FOR DETERMINING 
ENERGY STATE OF PLANTS 
Te May Ching 
Crop Science Department 
Oregon State University 
Cowallis, Oregon 
The bioluminescence produced by the luciferin-luciferase system is a very 
sensitive assay for ATP content in extracts of plant materials. With two 
additional enzymes, pyruvate b a s e  (E.C. 2.7.1.40) and adenylate kinase 
(E.C. 2.7.4.3), and an excess of phosphoenolpyruvate, the ADP and AMP in 
tissue extracts can be quantitatively converted to ATP and then assayed with 
the luciferase system (Ching and Ching, 1972). This simple, rapid, and sensi- 
tive procedure has been used for determining the energy state of plant mater- 
ials for metabolic studies (Ching and Ching, 1972; Ching et al., 1974) under 
different environmental stresses,* in cultivars of varied genetic capabilities 
(Ching and Kronstad, 1972), and in seeds of different viability and vigor 
(Ching, 1973; Ching and Danielson, 1972). The experimental results from 
our laboratory and others indicate that the bioluminescence test is a very 
useful tool for (a) predicting viability of seeds and pollens in a time period 
of one hour instead of the usual days or weeks required by growth tests; 
(b) screening cultivars for high growth potential and productivity without 
long-term and labor-demanding field tests; (c) detecting the incipient damage 
of environmental pollutants and stresses so that reparative measures can be 
implemented; and (d) discerning the role of ATP concentration, total con- 
tent of adenosine phosphates, ATP/ADP ratio, and energy charge [EC, EC = 
( [ATP] + 1 /2  [ADP] )/([ATPI + [ADP] + [AMP] ) - (7)] in metabolic con- 
trol of enzymes, organelles, tissues, and organisms. All these possibilities 
will be discussed in the following sections in more detail and particular 
applicational requirements will be mentioned. 
ATP TEST FOR SEED AND POLLEN VlAB!LlTV AND VIGOR 
ATP is needed for biosynthesis of nucleotides, nucleic acids, proteins, lipids, 
amino acids, and carbohydrates (Henderson and Paterson, 1973). ATP also 
modifies existing enzymes in cells and tissues, thus it activates, inactivates, 
- 
* Barlow, E. W. R., "Physiological Effects of Water Stress on Young C o ~ n  Plants," 
Ph.D. thesis, 1974, Oregon State University. 
https://ntrs.nasa.gov/search.jsp?R=19760008456 2020-03-22T18:05:46+00:00Z
or amplifies the enzyme activity, depending on the condition (Holzer and 
Duntze, 1972). In seeds as well as pollens, ATP is usually low and often 
limiting for the early metabolic events of germination (Ching, 1972; Oben- 
dorf and Marcus, 1974). If more ATP can be synthesized, the metabolic 
wheel will be turned faster, which eventually leads to more growth. Figure 1 
shows such correlation in rape seeds. Further indication of high ATP and 
energy charge associated with high synthetic ability of ribonucleic acid (RNA), 
protein, and lipids in wheat embryos or seedlings are summarized in table 1. 
In pollen of douglas fir (Pseudotsuga menziesii Franco), the ATP contents 
were found to  be 50, 136,209,292,456, and 562 n moles per one gram of 
fresh weight for lots having 0,9, 16,30,57, and 91 percent germination, 
respectively. Again, the data indicate a high correlation of ATP content 
and germinability (unpublished data from our laboratory). All these data 
show that among the seed or pollen lots of one crop or cultivar, ATP content 
appears to  be a good biochemical index of vigor and viability. In order to be 
of practical use for seed testing or cross pollination work, a standard curve 
depicting the relationship of ATP content and growth potential or germin- 
ability should be established for each cultivar of a crop. At present, several 
Figure 1.  Correlation of ATP content in 4-hour imbibed seeds of  rape 
(Brassica napus L.) and seed weight, 4day seedlings length, 4day  seedling 
fresh and dry weight. X, 0 = different lots; r = correlation coefficient; 
** = significant a t  1 percent level (Ching and Ching, 1973). 
Table 1 
Contents of ATP and Total Adenosine Phosphates (TAP), Energy Charge 
(EC), Protein, RNA, and Lipid Synthesizing Ability and Growth Rate 
of the Embryo or Seedling of Wheat Cultivars, Hyslop (C.I. 14565) 
and Yarnhill (C.I. 14563) (Ching, 1972) 
standard curves have been established for cultivars of lettuce seeds (Ching and 
Danielson, 1972). More standard curves for different crops are therefore 
needed for this test. 
Embryo dry weight, mg 
4-hours embryo : ATP, pmole 
TAP, pmole 
EC 
2-day seedling: ATP, pmole 
TAP, pmole 
EC 
~ ~ - ' ~ ~ - a m i n o  acid incorporation 
(cpm/5 seedlings): 30 hours old 
44 hours old 
~ - ~ ~ - u r i d i n e  ncorporation 
(cpm/5 seedlings): 20 hours old 
36 hours old 
U L - ~ ~ C - ~ I U C O S ~  incorporation 
into lipids (cpm/5 seedlings) : 
24 hours old 
48 hours old 
7-day seedlings: fresh weight mg 
dry weight mg 
PREDICTOR FOR HIGH GROWTH POTENTIAL AND PRODUCTIVITY 
IN NEW CROSSES AND SELECTIONS OF BREEDING MATERIALS 
The data in table 1 indicate that between cultivars of the same crop, differ- 
ences exist in synthetic ability, and that a positive correlation of hlgh ATP 
content, total adenosine phosphates, and energy charge with high synthetic 
ability is clearly demonstrated. A time curve showing the fluctuation of 
ATP, ADP, and AMP pools in the wheat embryo or seedling further depicts 
Hyslop 
1.19 + 0.13 
232 + 29 
504 * 56 
0.71 
2985 + 315 
4819 -+ 501 
0.81 
1526 rt 184 
4701 + 301 
1060 + 172 
2438 + 313 
141 5 rt 152 
3229 + 289 
157 + 1 2  
18 + 1 
Yamhill 
1.26 + 0.14 
290 +35  
620 rt 41 
0.72 
4800 rt 360 
6656 -+ 702 
0.86 
2138 -+ 329 
7626 + 5 13 
1259 5 140 
5897 rt 647 
1760+ 154 
6242 rt 344 
201 + 15 
21 + 1 
the dynamic aspect of energy metabolism in germinating seeds (figure 2). 
It shows clearly that three oscillations of synthesis and utilization of adenosine 
phosphates occurred in the wheat embryo and seedlings during the first 48 
hours of germination. Each dip on the curve coincides with major metabolic 
and morphological events. The first one associates with protein and RNA 
synthesis (Ching, 1972; Mazus' and Buchowicz, 1973); the second one fol- 
lows DNA synthesis (Ching and Kronstad, 1972) and root emergence; and the 
third dip relates with the shoot emergence and the myriad of synthetic ac- 
tivities that followed. One trend shows clearly that the cultivar YamhiU with 
higher growth potential had higher ATP utilization and synthesis ability than 
Hyslop (table 1). Furthermore, the rate of synthesis exceed-~ti~izzk-uon much 
more in Yamhill, resulting in a larger pool of ATP and ADP anu $higher 
energy charge in Yamhill seedling. The larger pool and high energy charge in 
turn facilitates more biosynthesis and faster growth which eventually could 
lead to more productivity under optimum conditions. This speculation was 
born out by a cooperative study with Dr. R. G. McDaniel at the University 
of Arizona on nine newly developed Mexican wheat cultivars. In the study 
we found that the efficiency of oxidative phosphorylation of isolated mito- 
chondria and the adenylate energy charge of germinating seedlings are posi- 
tively significantly correlated with grain yield (correlation coefficients of 
+0.729* and +0.687*, respectively). Therefore, we have concluded that both 
biochemical parameters appear to be useful tools for plant breeders to 
Hours of Germination 
Figure 2. Changes in the content of ATP, ADP, AMP, and total adenosine 
phosphates i n  the embryo and the seedling of Hyslop and Yamhill wheat 
seed germinated at 293 K (20" C) in the dark (Ching and Kronstad, 1972). 
* Significantly correlated at the 5 percent level. 
52 
screen for high producers at an early stage of a breeding program to reduce 
the time- and money-consuming field trials. More information is needed in 
the application of this concept in actual programs. 
INDICATOR FOR ENVIRONMENTAL QUALITY AND STRESSES 
Salts in soil reduced the growth and the ATP content in pea roots (Hasson- 
Porath and Paljakoff-Mayber, 1971). Cold temperature decreased ATP in 
cotton seedlings and germinating douglas fir seeds (Stewart and Guinn, 1969; 
Ching and Ching, 1973). Herbicides (for example, chloropropham) lowered 
the ATP level aad,inhibited RNA synthesis in soybean hypocotyls (Gruen- 
hagen and Morgbnd, 1971), and atmospheric pollutants (for example, ozone) 
reduced ATP content and energy charge in douglas fir seedlings." Water 
stress or drought conditions caused a reduction of ATP content in corn 
leavest and germinating douglas fir seeds (Ching and Ching, 1973). From 
all these reports, one may develop a procedure with which to monitor the 
environmental influences and estimate the degree of damage or benefit so that 
reparative measures can be implemented. 
STUDIES ON METABOLIC REGULATION 
The regulation of carbohydrate metabolism by adenine nucleotides was 
known in 1964. Subsequently, Atkinson developed the concept of energy 
charge as an overall measure of the energy state of cells (Atkinson, 1969). 
Based on experimental data, Atkinson and his colleagues observed that when 
the energy charge is greater than 0.5, ATP-utilizing systems increase their 
activities, and, at an energy charge lower than 0.5, ATP-regenerating sequences 
become dominant. Therefore, the energy charge could modulate biosynthesis 
in cells and tissues. 
In plant materials (Davies, 1973), information regarding the regulation by 
adenine nucleotides are very scanty. ATP and citrate inhibited phospho- 
fructo-kinase in carrot, corn scutellum, and peas, whereas ADP and AMP 
were relatively ineffective in modulating the response to ATP. The plant 
isocitrate dehydrogenase responded neither to individual adenosine phosphates 
nor to energy charge, but the decarboxylation of alpha oxoglutarate was 
stimulated by AMP in the mitochondria of pea and cauliflower. Recently, the 
first positive evidence of energy charge controlling the activity of 3-phospho- 
glyceric acid kinase from pea leaves was reported (Pacold and Anderson, 
1973). The ribulose-5-phosphate kinase of the same tissue, however, was 
-- 
* Tingey, D., National Ecol. Res. Lab., U.S. Environmental Protection Agency, 
Corvallis, Oregon, private communication. 
t Barlow, E. W. R., "Physiological Effects of Water Stress on Young Corn Plants," 
Ph.D. thesis, 1974, Oregon State University. 
not controlled by the energy charge. The difference is difficult to resolve 
and awaits future experimentation. 
The data in table 1 indeed indicate a modulation of synthesis by energy 
supply and energy charge. At different times of seed germination and matur- 
ation, such positive correlations do  not exist, however, particularly at the 
peak of cell division (Ching and Ching, 1972) and the peak of synthesis of 
nucleotides and nucleic acids (Ching et al., 1975) (figure 3). Apparently a 
temporal control of development overrides the energy charge regulation or 
simply because the end product inhibition does not occur in the biosynthetic 
pathways of nucleotides (Henderson and Paterson, 1973). In tissues with 
stabilized cell numbers, such as the gametophyte of ponderosa pine seeds, an 
increased ATP content, total adenosine phosphates, and energy charge 
preceded the biogenesis of enzymes and organelles and then all reduced in 
concert after the function of these enzymes and organelles was fulfded 
(Ching , 1970) (figures 4,s). 
It is clearly shown from this discussion that many regulatory mechanisms 
are operative in the complex systems of eukaryotes, but few are absolutely 
established yet. The bioluminescence assay of adenosine phosphates will 
definitely facilitate the exploration of these mechanisms. 
POD L ENG TiY fmmj 
Figure 3. Contents of ATP, total adenosine phosphates (AP), DNA and 
RNA, and energy charge in whole 8day-old pods of rape (Ching, et al., 
1974). 
DAYS 
Figure 4. Changes in the content of ATP, ADP, AMP, and 
total adenosine phosphates (upper) and changes in adenylate 
energy charge (-) and ATP concentration (---) (lower) in the 
gametophyte of germinating ponderosa pine seeds (Ching and 
Ching, 1972). 
Figure 5. Changes in protein content and activity of fumarase and iso- 
citrate lyase with germination days in fractions isolated by sucrose density 
gradient centrifugation of 10,000-g pellet from 250 gametophytes of ger- 
minating ponderosa pine seeds. M = mitochondria; P = mixture; G = glyox- 
ysomes (Ching, 1970). 
REFERENCES 
Atkinson, D. E., Annu. Rev. Microbiol., 23, 1969, p. 47. 
Ching, T. M., Plant Physiol., 46,1970, p. 475. 
Ching, T. M., in Seed Biology, II, T. T. Kozlowski, ed., Academic Press, 
1972, p. 103. 
Ching, T. M., Plant Physiol., 51, 1973, p. 400. 
Ching, T. M. and K. K. Ching, Plant Physibl., 50,1972, p. 536. 
Ching, T. M. and K. K. Ching, in International Symposium on Dormancy 
in Trees, Polish Academy of Sciences, Komik, 1973, p. 13. 
Ching, T. M. and R. Danielson, Proc. Assoc. Ofi Seed Analysts., 62, 1972, 
p. 116. 
Ching, T. M. and W. E. Kronstad, Oop Sci., 12, 1972, p. 785. 
Ching, T. M., D. L. Stamp, and J. M. Crane, Plant Physiol., 54, 1974. 
Davies, D. D., in Biosynthesis and its Control in Plants, B. V. Milborrow, ed., 
Academic Press, 1973, p. 2. 
Gruenhagen, R. D. and D. E. Moreland, Weed Sci., 19, 1971, p. 3 19. 
Hasson-Porath, E. and A. Paljakoff-Mayber, Plant Physiol., 47, 1971, p. 109. 
Henderson, J. F. and A. R. P. Paterson,Nucleotide Metabolism, Academic 
Press, 1973, p. 54. 
Holzer, H. and W. Duntze, in Biochemical Regulatory Mechanisms in 
Eukaryotic Cells, E. Kun and S. Grisolia, eds., Wiley-Interscience, 1972, 
p. 115. 
Mazus', B. and J. Buchowicz, Phytochem., 12, 1973, p. 271. 
Obendorf, R. L. and A. Marcus, PlantPhysiol., 53,1974, p. 779. 
Pacold, I. and L. E. Anderson, Biochem. Biophys. Res. Comm, 51,1973, 
p. 139. 
Stewart, J. M. and G. Guinn,Plant Physiol., 44,1969, p. 605. 


Bioluminescence for determining energy state of plants

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