Spontaneous ultra-weak photon emission (UPE; also known as biophoton emission at wavelength of 180-800 nm), in the order of 10-16 W/m2, has been detected in various living systems, including microbial, plant, and animal species [1, 2, 3]. UPE is putatively associated with oxidative metabolism, intra- and inter-cellular communication, photosynthesis, growth, cell division and apoptosis, and death [4, 5, 6]. The development of highly sensitive detectors has enabled new investigation of UPE in the life and medical sciences, and in agriculture [3,7]. For example, two-dimensional images of UPE can be captured using a highly sensitive charge-coupled device camera. This is an excellent non-invasive method of investigation for the fields of microbiological, plant, and medical research . In medical research, UPE is being studied as a diagnostic tool [8,9]. In the plant sciences, UPE has been used to investigate the responses of plants to lunisolar tides and wounding . In agriculture, UPE is being used to identify new types of agrochemicals that potentiate plants’ defenses, or to differentiate herbicide-resistant from herbicide-susceptible weeds .
Despite the above studies, relatively little is known about the molecular mechanisms underlying UPE. An earlier study showed that UPE in bull spermatozoa is associated with adenosine triphosphate (ATP) levels. The increase in photon emission due to lipid peroxidation highly correlated with increases in cell ATP levels induced by thermal stress .
Most studies of UPE have focused on its link to plant growth and development [12,13]. The biological role of UPE in plants is not well understood, although it may be generated from the relaxation of electronically excited species formed during oxidative metabolic processes [14, 15, 16]. UPE has been shown to be closely related to photosynthesis, lipid peroxidation, catabolism, free radical reactions, radiation effects, detoxification, carcinogenic effects, aging, and the death process .
Strawberry (Fragaria x ananassa Duch.) fruit is soft and juicy when ripe, with high nutritional value. It is a typical non-climacteric fruit in that it ripens without ethylene or dramatic changes in cellular respiration. Although there have been many studies of the ripening, softening, and decay of the strawberry fruit, there is little known of how these processes are related to cellular energy levels. Recent studies showed that post-harvest softening and decay of the fruit are regulated by energy level [18,19].
To the best of our knowledge, there has been no report on UPE conducted in strawberry fruit. This study was conducted to further our understanding of UPE in plants and to determine whether there is an association between UPE and cellular energy levels during ripening of the strawberry fruit. The data from this study should provide new insights into the association between UPE and fruit development and senescence, the source of UPE production in plants, and a better understanding of the role of UPE in plants.
2 Materials and Methods
2.1 Fresh fruits
The strawberry fruits (cv. ‘Hongyan’) used in this study were collected from a farmers’ cooperative greenhouse at 5 ripening stages, I-V: I, green; II, light red; III, half-red; IV, red; and V, ripe. Within 30 min of harvest, the fruits were measured for UPE and cut into pieces for frozen storage in liquid nitrogen at −196°C for subsequent analysis.
2.2 Stored fruits
For the post-harvest studies, fruits were harvested at stage IV (red), washed, air-dried, packed into zip bags, and stored at 25 ± 1°C. After storage of 1-5 days, the fruits were sampled for UPE and biochemical analysis in the same manner as the fresh fruits.
2.3 UPE measurements
UPE measurements were performed as described [20,21], with some modifications, using the UPE detection system BPCL-SH15-TGC (Rio Tinto Technology, Beijing, China), in accordance with the manufacturer’s instructions. Ten randomly-selected fruits were measured at each timepoint, and each fruit was measured 10 times. A 1 cm (diameter) × 1.5 cm (height) tissue sample was punched from each fruit and placed into a cup in a dark chamber of the instrument for UPE measurement. The machine was pre-warmed for 30 min before use. UPE at each timepoint was calculated based on 10 fruits, each measured 10 times.
2.4 Biochemical analysis
To determine ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) content of the fruits, one gram of each frozen fruit was analyzed using high performance liquid chromatography (ELITE Lachrom Pump L-2130 equipped with Hitachi UV-VIS detector L-2420, Hitachi, Japan). All samples were measured in 3 replications. A Hitachi liquid chromatography column (LaChrom-C18; 4.6 mm × 250 mm, 5 μm) was eluted with 35 mmol/L phosphate (NaH2PO4) buffer (pH 6.8) at a flow rate of 1.0 mL/min and column temperature of 30 °C.
Standards of the highest quality for ATP, ADP, and AMP (Sigma, USA) were used to draw the concentration curves. The energy charge was calculated as (ATP + 0.5 ADP) / (ATP + ADP + AMP) .
2.5 Statistical analysis
Data were statistically analyzed using SPSS software and expressed as mean ± standard error.
The conducted research is not related to either human or animals use.
3.1 Changes in energy levels during fruit ripening
The levels of ATP, ADP, and AMP in the ripening strawberry fruits were measured (Figure 1). The data showed that ATP dropped quickly from stage I (0.89 mg/g) to the lowest at stage IV, and increased slightly afterward to 0.43 mg/g. ADP content was relatively low (about 0.22 mg/g) and stable before stage IV, and more than doubled at stage V. AMP was barely detectable and declined in the fruits over the entire ripening period. The energy charge from the three molecules (ATP, ADP, and AMP) varied between 0.75-0.92, and decreased as the fruit ripened (Figure 2).
3.2 Changes in UPE during fruit maturation
UPE during fruit ripening decreased almost linearly from stage I (63 count/s) to stage V (32 counts/s; Figure 3). The reduction was most remarkable between stages III and V.
3.3 Association between UPE intensity and energy in ripening fruits
Statistical analyses showed that there were linear correlations between the intensity of UPE (y) and energy charge (x), represented as AMP, ADP, or ATP levels (Table 1). Among them, ADP levels negatively correlated with UPE, while the others positively correlated.
The correlation analysis of UPE intensity (y) and energy level (x) in ripening strawberry fruits
|UPE regression equation||r*|
|AMP||y = 3889.961 x1 + 30.1172||0.9539|
|Energy charge||y = 204.886 x2 – 125.343||0.9500|
|ATP||y = 44.109 x3 + 25.0295||0.8004|
|ADP||y = –71.692 x4 + 63.4568||–0.6302|
3.4 Change in energy levels in post-harvest fruit
We then analyzed the energy changes after storage at room temperature in fruits harvested at stage V. The data showed that ATP content declined over the storage period, particularly 3 days after harvest, from 0.62 mg/g on day 1 to 0.13 mg/g on day 5. For ADP, the reduction was slow from 0.30 mg/g (day 1) to 0.22 mg/g (day 3), and it slightly increased after day 3 to 0.35 mg/g on day 5. AMP was barely detectable and the content increased from 0.01 mg/g on day 1 to 0.06 mg/g on day 5 (Figure 4). The energy charge from the 3 molecules declined from 0.85 (on day 1) to 0.57 (on day 5) during the period (Figure 5).
3.5 Change in UPE during post-harvest storage period
During the storage period, fruits continued to ripen and soften. Measurements showed that UPE increased slightly between days 1 and 2, and declined constantly afterward from 46 count/s (day 1) to 29 count/s (day 5; Figure 6).
3.6 Association between UPE intensity and energy during the post-harvest period
Regression analyses showed that there were linear correlations between the intensity of UPE (y) and energy charge (x) represented as AMP, ADP, or ATP levels (Table 2). Among them, ADP and AMP levels negatively correlated with UPE, while ATP and energy charge positively correlated.
The correlation analysis of UPE intensity (y) and energy level (x) in ripe strawberry fruits during post-harvest storage period
|UPE regression equation||r*|
|Energy source||y = 72.327 x1 – 13.6758||0.9807|
|ATP||y = 36.827 x2 + 26.0633||0.9403|
|AMP||y = –417.633 x3 + 50.6589||–0.9457|
|ADP||y = –91.535 x4 + 64.6953||–0.6433|
Strawberry is a typical non-climacteric fruit. Many studies of the physiology of this important fruit have shown that during ripening numerous changes occur at the gene and molecular levels. However, to our knowledge, UPE has not investigated in the fruit. Using a highly sensitive detector, we were able to detect UPE in the fruit during ripening and the post-harvest storage period. Our data showed that UPE declines during the ripening stages, and we further found that this decline is concurrent with decline in cellular energy levels.
The association between UPE and plant development is largely unclear, and how UPE is generated in the plant is still an open question. Tang et al.  speculated that UPE resulted from energy released as photons from ATP. In their study with Chinese cabbage, Li et al.  proposed that UPE is derived from the electron transfer chain during photosynthesis. In an in vitro study of UPE with mitochondria, it was shown that the intensity of UPE in mitochondrial extract was positively related to the mitochondrial concentration . Mitochondria and chloroplasts are the main organelles in which oxidation and energy conversion take place. These results suggest that UPE may originate from subcellular organelles such as mitochondria and chloroplasts.
Furthermore, a study showed that, during flowering, UPE from the apricot flower was concurrent with changes in ATP levels , suggesting that ATP and energy levels are likely associated with UPE. In the present experiment, we found that UPE intensity decreased gradually during the maturation of strawberry fruit, and was significantly and positively related to cellular ATP and energy charge levels. Thus, it appears likely that ATP is the energy source for UPE. This is consistent with earlier observations [11,18].
The associations between UPE and the other 2 energy molecules, ADP and AMP, were not consistent during the ripening and post-harvest storage period (Table 1 and 2). This might be because the metabolic processes of ATP, which is the precursor of ADP and AMP, during the two periods are different. Furthermore, measurements showed that the AMP content was very low (Figure 1 and 4), and may therefore contribute very little to UPE. The negative association between ADP and UPE levels suggests that conversion of ATP to ADP reduces the cellular energy charge effective for UPE. Therefore, the levels of ATP and energy charge may be the main determinant of UPE level. Although the energy charge is based on the ATP, ADP, and AMP contents, our study showed that during the fruit ripening and post-harvest period, most of the contribution to energy charge is from ATP. This is consistent with previous work .
Earlier studies showed that the generation of active oxygen species can accelerate decay of the fruit , and active oxygen production is related to NAD(H) and NADP(H) . A more recent study showed that storage of fruit in pure oxygen increased respiration and the generation of ATP, which helps to maintain the integrity of the cell membrane and delay fruit decay . Further study is needed to determine how other energy carriers and molecules are involved in UPE and whether UPE can be used for profiling the quality or other agronomic traits of the fruit.
UPE from ripening strawberry fruits declined during ripening and the post-harvest period, and the decline is concurrent with the decline in cellular ATP and energy charge levels. The significant and positive correlations between the two parameters suggest that ATP is the main source of energy for UPE in the fruit.
Amano T., Kobayashi M., Devaraj B., Usa M., Inaba H., Ultraweak biophoton emission imaging of transplanted bladder cancer, Urol. Res., 1995, 23, 315-318
Gallep C.M., Moraes T.A., Cervinkova K., Cifra M., Katsumata M., Barlow P.W., Lunisolar tidal synchronism with biophoton emission during intercontinental wheat-seedling germination tests, Plant signaling & behavior, 2014, 9, e28671
Kobayashi M., Highly sensitive imaging for ultra-weak photon emission from living organisms, Journal of photochemistry and photobiology B, Biology, 2014,139, 34-38
Prasad A., Rossi C., Lamponi S., Pospisil P., Foletti A., New perspective in cell communication: potential role of ultra-weak photon emission, Journal of photochemistry and photobiology B, Biology, 2014, 139, 47-53
Pospisil P., Prasad A., Rac M., Role of reactive oxygen species in ultra-weak photon emission in biological systems, Journal of photochemistry and photobiology B, Biology, 2014, 139, 11-23
Rastogi A., Pospisil P., Ultra-weak photon emission as a non-invasive tool for the measurement of oxidative stress induced by UVA radiation in Arabidopsis thaliana, Journal of photochemistry and photobiology B, Biology, 2013, 123, 59-64
Flor-Henry M., McCabe T.C., de Bruxelles G.L., Roberts M.R., Use of a highly sensitive two-dimensional luminescence imaging system to monitor endogenous bioluminescence in plant leaves, BMC plant biology, 2004, 4, 19
Yang M., Pang J., Liu J., Liu Y., Fan H., Han J., Spectral discrimination between healthy people and cold patients using spontaneous photon emission, Biomedical optics express, 2015, 6, 1331-1339
Chen P., Zhang L., Zhang F., Liu J.-T., Bai H., Tang G.-Q., et al., Spectral discrimination between normal and leukemic human sera using delayed luminescence, Biom. Opt. Exp., 2012, 3, 1787-1792
Kato K., Iyozumi H., Kageyama C., Inagaki H., Yamaguchi A., Nukui H., Application of ultra-weak photon emission measurements in agriculture, Journal of photochemistry and photobiology B, Biology, 2014, 139, 54-62
Guminska M., Kedryna T., Laszczka A., Godlewski M., Slawinski J., Szczesniak-Fabianczyk B., et al., Changes in ATP level and iron-induced ultra-weak photon emission in bull spermatozoa, caused by membrane peroxidation during thermal stress, Acta biochimica Polonica, 1997, 44, 131-138
Hou X., Liao X., Li Y., Zhang X., Bu W., Jia Y., et al., Ultraweak biophoton emission and its mechanism during seed germination of amaranthus hypochondriacus, Seed, 2004, 23, 23-29
Liu H., Liao X., Wu L., Jiang J., Effect of heat shock on biophoton and activities of antioxidant enzymes in immature wheat grains, J. Food Sci. Biotech., 2006, 25, 75-78
Van Wijk R., Van Wijk E.P.A., Wiegant F.A.C., Ives J., Free radicals and low-level photon emission in human pathogenesis: state of the art, Ind. J. Exp. Bio., 2008, 46, 273-309
Rastogi A., Pospisil P., Spontaneous ultraweak photon emission imaging of oxidative metabolic processes in human skin: effect of molecular oxygen and antioxidant defense system, J. Biomed. Opt., 2011, 16, 096005
Slawinski J., Biophotons from stressed and dying organisms: toxicological aspects, Indian J. Exp. Bio., 2003, 41, 483-493
Chang J.J., Physical properties of biophotons and their biological functions, Ind. J. Exp. Biol., 2008, 46, 371-377
Saquet A., Streif J., Bangerth F., Changes in ATP, ADP and pyridine nucleotide levels related to the incidence of physiological disorders in Conference pears and Jonagold apples during controlled atmosphere storage, J. Horticul. Sci. Biotech., 2000, 75, 243-249
Kan J., Wang H., Jin C., Huang I., Changes of active oxygen and mitochondria respiratory metabolism-related enzymes during maturation of peach fruit, Food Sci., 2009, 30, 275-279
Zhang X., Li F., Yang H., Wang F., The relationship between ultraweak luminescence and respiratory climacteric during ripening process in peach fruits, Acta Chin. Soc. Agr. Mach., 2004, 35, 215-217
Zhao D., Sheng J., Ding Y., Shen L., Fan B., Liu C., Nondestructive examination of tomato chilling injury by ultraweak luminescence., Spect. Spect. Anal., 2010, 30, 2493-52495
Li H., Men Q., Xia K., Liang Z., Ning K., Modern plant physiology Beijin Higher Education Press; 2002
Tan S., Xing D., Tang Y., Li D., Spectral studies of ultra-weak biophoton emission from plant’s leaves., Act. Photo. Sin., 2000, 29, 961-965
Li D., Tang Y., He Y., Xin D., The Study on mechanism of ultraweak luminescence of chloroplast in Chinese cabbage, Acta Laser Biology Sinica, 2002, 11, 64-66
Zhang X., Yang H., Ultraweak bioluminescence of chloroplast and mitochondria in plants, Plant Phy. Comm., 2004, 40, 111-114
Zhang X., Yang H., Li F., Zhang W., Changes in ultraweak luminescence, ATP and active oxygen contents during apricot florescence, J. Plant Phy. Mol. Biol., 2004, 30, 41-44
Chen J., Jin P., Li H., Cai Y., Zhao Y., Zheng Y., Effects of low temperature storage on chilling injury and energy status in peach fruit Tran CSAE 2012, 28, 4, 275-280
Chen X., Zheng Y., Yang Z., Ma S., Feng L., Wang X, Effects of high oxygen treatments on active oxygen metabolism and fruit decay in postharvest strawberry, J. Nanjing Agricultural University, 2005, 28, 99-102
Gu C., Zhu D., Li Q., Relationship between NAD Kinase and NAD(H), NADP(H) andactive oxygen during ripening and senescence of postharvested strawberry fruit, Sci. Agr. Sin., 2007, 40, 352-357
Liu T., Qian Z., Yang E., Wu F., Qu H., Jiang Y., Respiratory activity and energy metabolism of harvested litchi fruit and their relationship to quality deterioration, J. Fruit. Sci., 2010, 27, 946-951