JP4813327B2 - Photosynthesis sample evaluation method and photosynthesis sample evaluation program - Google Patents

Description

  The present invention relates to a photosynthesis sample evaluation method and a photosynthesis sample evaluation program.

  2. Description of the Related Art Conventionally, as a method for evaluating the influence of unknown chemical substances existing in the environment on living organisms, a biological toxicity test “bioassay” for examining growth inhibition of living organisms such as bacteria and algae has been used. As one of the bioassays, a technique using delayed fluorescence is known. For example, the following Non-Patent Document 1 describes that the light emission pattern of delayed light emission changes between a sample left for 2 hours in the dark before the delayed light emission measurement and a sample that is not left standing, and the wavelength of light irradiated before the delayed light emission measurement. It is disclosed that the light emission pattern of delayed light emission changes when the value is changed.

Patent Document 1 below discloses a method for evaluating harmful substances using delayed fluorescence. In this harmful substance evaluation method, first, the amount of delayed fluorescence (delayed luminescence) from an aqueous solution sample containing a toxic substance and the amount of delayed fluorescence from a comparative sample are respectively measured. Then, based on the measured amount of delayed fluorescence, the elapsed time at the characteristic point of the temporal change in the amount of delayed fluorescence is calculated, and a comparison value of the elapsed time is obtained to obtain a harmful substance present in the aqueous solution sample. To evaluate. Also in the following Patent Document 1, it is disclosed that the pattern of delayed light emission changes when the light intensity or the light wavelength of light applied to an object is changed.
International Publication No. 2005/062027 Pamphlet Werner Schmidt and Horst Senger, “Long-term delayed luminescence in Scenedesmus obliquus II Influence of exogeneous factors”, Biochimica et Biophysica Acta, Elsevier Science Publishers, 1987, 891 (1987), p.22-27

  However, in the conventional evaluation method described above, the influence on the reaction process of the photosynthetic function of the photosynthetic sample is not properly evaluated. That is, even if the influence of the photosynthetic sample on the entire photosynthetic function is known, it is difficult to evaluate the influence on each reaction process.

  The present invention has been made to solve the above-described problems, and provides a photosynthetic sample evaluation method and a photosynthetic sample evaluation program capable of appropriately evaluating the photosynthetic function of a photosynthetic sample included in an evaluation sample. For the purpose.

  The inventor of the present invention generally describes the light emission amount of delayed luminescence (delayed fluorescence: hereinafter referred to as delayed luminescence) from the photosynthetic sample, which increases as the amount of excitation light applied to the photosynthetic sample increases. I got a good knowledge. That is, the inventor of the present invention relates to the change over time in the amount of delayed luminescence, when the light synthesis sample is irradiated with intense light, while the amount of luminescence in a certain measurement time period decreases, The knowledge that the amount of luminescence increases was obtained. Furthermore, the inventors of the present invention have found that the increase or decrease in the amount of delayed luminescence is caused by a change in the redox state of the photosynthetic electron transport system of the photosynthetic sample (hereinafter referred to as “the redox state of the photosynthetic sample”) due to strong light. It was found that the movement of molecules in the photosynthetic electron transport system and changes in the distribution of electrons are reflected.

  Therefore, in order to solve the above problems based on the above-mentioned knowledge, the photosynthesis sample evaluation method of the present invention is a photosynthesis sample evaluation method for evaluating an evaluation sample including a photosynthesis sample having a photosynthesis function. A first excitation step of irradiating the first excitation light under a predetermined first irradiation condition, and after the first excitation step, the second excitation light is applied to the evaluation sample by comparing the first irradiation condition with the first irradiation condition. A second excitation step for irradiation under a second irradiation condition having a small value, a measurement step for measuring the amount of delayed emission generated from the evaluation sample after the second excitation step, and the delayed emission measured in the measurement step. And an evaluation step for deriving an evaluation value based on the light emission amount and evaluating the evaluation sample based on the evaluation value and the standard data. In the measurement step, the second irradiation condition is changed to change the first excitation. The amount of luminescence after the step and the second excitation step is measured, and in the evaluation step, the evaluation sample is evaluated based on the evaluation value corresponding to the luminescence amount measured in the measurement step and the standard data corresponding to the evaluation value. It is characterized by

  The photosynthesis sample evaluation program of the present invention is a photosynthesis sample evaluation program for evaluating an evaluation sample including a photosynthesis sample having a photosynthesis function. The computer uses a computer to output a first excitation light to a predetermined first sample. After the first excitation means and the first excitation means, the second excitation light is irradiated to the evaluation sample under the second irradiation condition having a smaller integrated light energy than the first irradiation condition. An evaluation value is derived based on the second excitation means, the measurement means for measuring the amount of delayed luminescence emitted from the evaluation sample after the second excitation means, and the amount of delayed luminescence measured by the measurement means And functioning as an evaluation means for evaluating the evaluation sample based on the evaluation value and the standard data, and the measurement means changes the second irradiation condition and measures the amount of light emitted after the first excitation means and the second excitation means. And evaluate Stage, and evaluating the evaluation sample on the basis of the standard data corresponding to the evaluation value and the evaluation value corresponding to the light emission amount measured by the measuring means

  According to the photosynthesis sample evaluation method and the photosynthesis sample evaluation program, first, the first excitation light is irradiated to the evaluation sample. Thereafter, the second excitation light is irradiated on the evaluation sample under irradiation conditions set so that the energy integrated value is smaller than that of the first excitation light. When the irradiation of the second excitation light is completed, the amount of delayed emission from the evaluation sample is measured. A plurality of such delayed emission amounts can be obtained by changing the second irradiation condition. Then, an evaluation value corresponding to the measured light emission amount is derived, and the evaluation sample is evaluated based on the evaluation value and the standard data. As described above, since the light emission amount of delayed luminescence can be obtained for each different second irradiation condition for the evaluation sample, the influence on the case where the redox state of the photosynthetic sample is different can be derived. It is possible to appropriately evaluate the photosynthetic function of the photosynthetic sample included in the evaluation sample.

  In addition, after irradiation with the first excitation light, the amount of delayed emission is measured after irradiation with the second excitation light so that the energy integrated value is lower than that of the first excitation light. Therefore, it is possible to make the measurement conditions for the amount of delayed light emission constant, and as a result, the amount of light emission of delayed light emission can be measured with higher accuracy. The standard data is data used as a reference when evaluating the evaluation sample, and the energy integrated value refers to the light energy integrated value for the photosynthetic sample.

  In the photosynthetic sample evaluation method of the present invention, the standard data is a reference value, and the evaluation step is based on an evaluation value corresponding to the light emission amount measured in the measurement step and a reference value corresponding to the evaluation value. It is preferable to evaluate the evaluation sample. In this case, by evaluating the evaluation sample based on the reference value, it is possible to appropriately evaluate the photosynthesis function of the photosynthesis sample included in the evaluation sample.

  In the photosynthetic sample evaluation method of the present invention, it is preferable that the evaluation step derives an evaluation value based on an integrated value obtained by integrating the light emission amounts of delayed luminescence measured in the measurement step every one or more measurement time zones.

  In this case, an integrated value of the amount of delayed light emission in a certain measurement time zone is calculated, and an evaluation value is derived based on the integrated value. By using such an integrated value, it is possible to easily and appropriately extract the redox state of the photosynthetic sample in a certain measurement time period, which appears in the temporal change in the amount of delayed luminescence. As a result, the photosynthetic function of the photosynthetic sample included in the evaluation sample can be more appropriately evaluated. In addition, when a plurality of measurement time zones are provided and the integrated value of each measurement time zone is calculated, it is possible to appropriately grasp the temporal change in the amount of delayed light emission. Therefore, it is possible to appropriately evaluate the photosynthetic function of the photosynthetic sample included in the evaluation sample by using the temporal changes over the plurality of measurement time zones.

  In the photosynthetic sample evaluation method of the present invention, the evaluation step sets the coefficient values of the plurality of functions so that the time-dependent change in the amount of delayed luminescence measured in the measurement step is fitted as a sum of a plurality of preset functions. It is preferable to derive the evaluation value based on the coefficient value.

  In this case, coefficient values of the plurality of functions are derived such that the temporal change in the amount of delayed light emission is fitted as a sum of a plurality of preset functions, and the evaluation value is derived based on the coefficient values. An evaluation value can be objectively derived by using such a mathematical method. In addition, since the time-dependent change in the amount of delayed light emission is fitted to the sum of a plurality of functions, evaluation based on the mechanism of delayed light emission is possible. Therefore, the photosynthetic function of the photosynthetic sample included in the evaluation sample can be appropriately evaluated.

  In the photosynthetic sample evaluation method of the present invention, it is preferable that the second irradiation condition is a waiting time from the end of irradiation of the first excitation light to the start of irradiation of the second excitation light.

  In this case, processing from the irradiation of the first excitation light to the measurement of the amount of delayed emission is performed by changing the irradiation start time of the second excitation light. When to start irradiation with the second excitation light (how much standby time is provided) is closely related to a change in the redox state of the photosynthetic sample. Therefore, it is possible to appropriately evaluate the photosynthetic function of the photosynthetic sample included in the evaluation sample by deriving an evaluation value based on the light emission amount of delayed emission obtained by changing the irradiation start timing of the second excitation light. it can. Further, in order to change the second irradiation condition, it is sufficient to change the waiting time, and it is not necessary to change the excitation light itself. Therefore, it is possible to easily set a situation where the redox state of the photosynthetic sample is different.

  In the method for evaluating a photosynthetic sample of the present invention, it is preferable that the second irradiation condition is at least one of the light amount, wavelength, pulse width, and irradiation time of the second excitation light.

  In this case, processing from the irradiation of the first excitation light to the measurement of the amount of delayed emission is performed by changing at least one of the light amount, wavelength, pulse width, and irradiation time of the second excitation light. These changed conditions are closely related to changes in the redox state of the photosynthetic sample. Therefore, by deriving an evaluation value based on the amount of delayed emission obtained by changing at least one of these elements, the photosynthetic function of the photosynthetic sample included in the evaluation sample can be appropriately evaluated. .

  In the photosynthetic sample evaluation method of the present invention, it is preferable that the measurement step measures the amount of light emitted after repeating the first excitation step and the second excitation step a plurality of times while changing the second irradiation condition.

  In this case, the processing from the irradiation of the first excitation light to the measurement of the light emission amount of delayed light emission is repeated a plurality of times while changing the second irradiation condition. Then, an evaluation value corresponding to each measured light emission amount is derived, and the evaluation sample is evaluated based on the evaluation value and the standard data. In this way, the photosynthesis sample included in the evaluation sample is not affected by external factors by measuring the amount of delayed luminescence multiple times while changing the second irradiation condition within a relatively short time. It is possible to appropriately evaluate the photosynthetic function.

  In the photosynthesis sample evaluation method of the present invention, it is preferable that the evaluation step evaluates environmental factors given to the evaluation sample. In this case, the influence of environmental factors on the photosynthetic sample in the evaluation sample can be appropriately evaluated.

  In the method for evaluating a photosynthetic sample of the present invention, the environmental factor is preferably a harmful substance. In this case, the influence of harmful substances on the photosynthetic sample in the evaluation sample can be appropriately evaluated.

  In the photosynthetic sample evaluation method of the present invention, the environmental factor is preferably one that inhibits the photosynthetic reaction of the photosynthetic sample. In this case, the mechanism by which environmental factors inhibit the photosynthetic reaction of the photosynthetic sample can be appropriately evaluated.

  In the photosynthetic sample evaluation method of the present invention, it is preferable that the evaluation step evaluates the efficiency of photosynthetic electron transfer of the photosynthetic sample. In this case, the efficiency of photosynthetic electron transfer of the photosynthetic sample can be appropriately evaluated.

  In the photosynthesis sample evaluation method of the present invention, it is preferable that the evaluation step evaluates the growth state of the photosynthesis sample. In this case, it is possible to appropriately evaluate the growth state of the photosynthetic sample, thereby contributing to the growth diagnosis of the plant.

  In the photosynthesis sample evaluation method of the present invention, it is preferable that the evaluation step evaluates the function of the agricultural chemical given to the evaluation sample. In this case, the function of the agricultural chemical can be appropriately evaluated.

  According to the photosynthesis sample evaluation method and the photosynthesis sample evaluation program, the photosynthesis function of the photosynthesis sample included in the evaluation sample can be appropriately evaluated.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same or equivalent elements are denoted by the same reference numerals, and redundant description is omitted.

(Theoretical background)
First, with reference to FIG. 1, a description will be given of photosynthetic electron transfer, which is the theoretical background of the photosynthetic sample evaluation method of the present invention. FIG. 1 is a schematic diagram showing photosynthetic electron transfer.

  In general, when a photosynthesis sample having a photosynthetic function absorbs a large amount of light energy, rate limiting occurs in individual reactions of the photosynthetic electron transfer system, and thus photosynthesis electron transfer does not proceed smoothly. In other words, the redox state of the photosynthetic sample becomes different from the normal state. Such a state can be obtained by changing the light conditions applied to the photosynthetic sample. Here, the light conditions are, for example, the light amount, wavelength, pulse width, and irradiation time of light given to the photosynthetic sample. In the present specification, a state in which photosynthetic electron transfer does not proceed smoothly (a state in which the redox state of the photosynthetic sample is different from normal) is referred to as “saturation” of the photosynthetic electron transfer system. On the other hand, a state in which photosynthetic electron transfer proceeds smoothly (the redox state of the photosynthetic sample is a normal state) is referred to as “no saturation”.

As shown in FIG. 1, the photosynthetic reaction of the photosynthetic sample mainly consists of a photosystem II complex (PSII), cytochrome b6f complex (cytb ₆ f), and photosystem I complex (PSI) present in the chloroplast thylakoid membrane. ) And a reducing substance (NADPH) produced by ATP synthase and a carbon dioxide fixation reaction (Calvin-Benson cycle) using ATP synthase.

First, PSII and PSI absorb light and perform an electron transfer reaction. Specifically, when irradiated with light, water molecules (H ₂ O) are decomposed in PSII, and electrons (e ⁻ ) are taken out. This electron is transferred from the PSII reaction center (P680) via the Q _A electron acceptor (Q _A ) to the oxidized plastoquinone (pQ _OX ) bound to the Q _B electron acceptor (Q _B ) site. . This produces reduced plastoquinone (pQ _red ).

Reduction plastoquinones, after moving to the free to plastoquinone pool (pQpool) from _{Q B} site, is reoxidized by Cytb ₆ f, returns to oxidized plastoquinone. At this time, hydrogen ions (H ⁺ ) flow into the thylakoid membrane through cytb ₆ f to form a proton gradient. Hydrogen ions that form a proton gradient are used for ATP synthesis by ATP synthase. On the other hand, electrons extracted during reoxidation of reduced plastoquinone are transferred to the PSI reaction center (P700). This electron is transferred from P700 to ferredoxin (Fd) in PSI and contributes to the production of NADPH. Excess electrons after PSI (reducing power) are transferred to cytb ₆ f via Fd by circulating electron transfer, and reduce oxidized plastoquinone. This results in reduced plastoquinone (cyclic electron transfer).

  Delayed luminescence is considered to be mainly caused by chemical re-excitation of the reaction center chlorophyll of PSII as a result of the reverse reaction of the above-mentioned photosynthetic electron transfer reaction, but PSI is also involved in luminescence as well. There is also a report that it is doing. Further, it is understood that electrons distributed inside PSII, pQpool, and after PSI are main components contributing to chlorophyll reexcitation. Therefore, when the photosynthetic electron transfer reaction changes due to the influence of environmental factors (for example, harmful substances), a change appears in delayed luminescence.

In general, the electron transfer capability of pQpool, cytb ₆ f, and PSI is lower than that of PSII. Therefore, when PSII is excessively reduced by light irradiation, pQpool is excessively reduced by electron transfer from PSII, so that the oxidized plastoquinone that receives electrons from PSII decreases, and the efficiency of photosynthetic electron transfer from PSII. Is known to decrease. Such a phenomenon is one of the saturation of photosynthetic electron transfer. More specifically, when PSII excessively absorbs light, first, reoxidation of reduced plastoquinone by cytb ₆ f becomes rate-determining, and there is a lack of oxidized plastoquinone that can accept electrons from PSII. . Therefore, the electron transfer from PSII to cytb ₆ f is stagnant. As a result, the electrons are accumulated excessively in PSII, and the reaction center of PSII is destroyed. Such an effect is called photoinhibition.

  In order to avoid such photoinhibition, it is known that the light-collecting chromoprotein complex (LHC2) originally for supplying electrons to PSII causes various metabolic changes such as transfer to PSI (state transition). It has been. The metabolic change caused by such light inhibition is gradually recovered by stopping the excessive light irradiation.

  The present invention evaluates the environmental factors given to the photosynthetic sample by paying attention to the change in the redox state of the photosynthetic electron transfer as described above and measuring delayed emission by controlling the irradiation of the excitation light. .

(Delayed luminescence measuring device)
First, a delayed luminescence measuring apparatus for carrying out the photosynthetic sample evaluation method according to the present invention will be described with reference to FIGS. FIG. 2 is a diagram schematically illustrating the delayed light emission measuring device 1 according to the embodiment. FIG. 3 is a block diagram partially showing the delayed light emission measuring device 1 shown in FIG.

  The delayed light emission measurement device 1 includes a measurement unit 10, an analysis unit 20, and a control unit 30. The measurement unit 10 and the analysis unit 20 are connected by a first cable 41, and the analysis unit 20 and the control unit 30 are connected by a second cable 42.

  The measurement unit 10 includes a sample placement unit 11, a light source 12, a filter 13, a condensing optical system 14, a shutter 15, and a detection unit 16, and these components are housed in a housing 17. The housing 17 includes a main body portion 17a and a lid portion 17b.

  The sample placement unit 11 is a part for placing a sample to be measured. The sample installation unit 11 is configured so that a container containing a sample can be installed, and the sample can be exchanged by opening the lid 17b. In addition, the sample installation part 11 may be comprised so that a sample can be introduce | transduced from the exterior of the housing | casing 17 with a liquid feeding pump.

  The light source 12 irradiates the sample installed in the sample installation unit 11 with light (excitation light). The light source 12 should just be able to radiate | emit the light wavelength (280-800 nm) effective for photosynthesis of a plant, for example, a light emitting diode, a semiconductor laser element, or a light bulb is used. The light source 12 may be a monochromatic light source or a light source that combines a plurality of light sources. The light emission method of the light source 12 is not limited. For example, a method of continuously emitting light for a predetermined time, a method of pulse lighting with an arbitrary pattern, a method of sequentially emitting a plurality of light sources having the same or different wavelength characteristics, A method of simultaneously emitting light from a light source is conceivable.

  The filter 13 transmits delayed light emission, and is provided in contact with the inner wall surface of the housing 17.

  The condensing optical system 14 condenses the weak delayed light that has passed through the filter 13. The condensing optical system 14 is provided so that the delayed light emission condensed thereby proceeds to the detection unit 16.

  The shutter 15 is provided so as to be freely opened and closed. When the shutter 15 is closed, delayed light emission from the condensing optical system 14 to the detection unit 16 is blocked. The shutter 15 may be provided at a position closer to the light source 12 than the filter 13 and the condensing optical system 14. In this case, the light emission (afterglow) of components, such as a condensing optical system, by the light from the light source 12 directly entering into the filter 13 and the condensing optical system 14 can be prevented.

  The detection unit 16 measures the amount of delayed luminescence generated from the photosynthesis sample in the sample by the light emitted from the light source 12, and transmits the measurement result to the analysis unit 20. The detection unit 16 includes an optical sensor 16a that detects delayed emission, and a light amount calculation unit 16b that calculates the amount of delayed emission based on a signal detected by the optical sensor 16a. The detection unit 16 includes, for example, a photon counter using an electron multiplier, a faint light measuring device using an avalanche photodiode, and the like.

  The analysis unit 20 includes a recording unit 21, a calculation unit 22, a display unit 23, and an input unit 24. The recording unit 21 records the measurement result transmitted from the detection unit 16 and data necessary for analysis or evaluation. The calculation unit 22 performs calculation analysis and evaluation based on the measurement results and data recorded in the recording unit 21. The display unit 23 displays the result analyzed and evaluated by the calculation unit 22. The input unit 24 is an interface through which data necessary for analysis can be input.

  The control unit 30 can control operations of the measurement unit 10 and the analysis unit 20, and is configured by combining a computer, a timer, a relay, and the like, for example. In particular, the control unit 30 includes a light source control unit 31 and a detection control unit 32. The light source control unit 31 can control the light conditions (for example, irradiation timing, light amount, wavelength, pulse width, and irradiation time) of the excitation light emitted from the light source 12. The detection control unit 32 can control the detection (measurement) of the amount of delayed light emission in a predetermined time. For example, the detection control unit 32 controls the detection time, the detection time, and the like.

  In addition, the structure of the analysis unit 20 and the control unit 30 is not limited to this, For example, one computer may have the function of the analysis unit 20 and the control unit 30 together.

(Method for evaluating photosynthetic samples)
Next, the procedure of the photosynthetic sample evaluation method performed by the delayed luminescence measuring apparatus 1 will be described with reference to FIG. FIG. 4 is a flowchart showing the procedure of the photosynthesis sample evaluation method.

  First, two types of samples including a photosynthesis sample are prepared. One of these is a standard sample in which the conditions such as the amount of the photosynthetic sample, the light amount or wavelength of the light applied to the photosynthetic sample, the temperature, the composition of the buffer solution, and the exposure of the chemical substance are set to predetermined conditions (step S11b). ). The other is an evaluation sample in which at least one of environmental conditions (addition of harmful substances, temperature, etc.) or conditions of the photosynthetic sample itself (growth conditions, etc.) differ from the standard sample (step S11a). The prepared standard sample and evaluation sample are stored in a container such as a cuvette, and left as it is under controlled conditions of temperature, light, etc. as necessary until the effect of the comparison conditions on the photosynthetic sample sufficiently appears. Incubate.

  Subsequently, the first excitation light is irradiated under a predetermined first irradiation condition to the evaluation sample that has sufficiently exerted the influence of the comparison condition on the photosynthetic sample (step S12a, first excitation step). The light source control unit 31 outputs a control signal to the light source 12 so that the excitation light emitted from the light source 12 satisfies the first irradiation condition. The first irradiation condition is to irradiate light having a predetermined light amount and a predetermined wavelength so that the photosynthetic electron transfer performed in the photosynthetic sample is saturated for a predetermined time. Excitation light emitted from the light source 12 reaches a container installed in the sample installation unit 11. Such irradiation of the first excitation light is similarly performed on the standard sample (step S12b, first excitation step).

  Subsequently, the evaluation sample irradiated with the first excitation light is kept on standby for a predetermined time (0 seconds or more) in a predetermined light condition (step S13a, second excitation step). Specifically, the evaluation sample is put on standby in an environment where the light energy of the light applied to the evaluation sample is smaller than that of the second excitation light described later. For example, light is prevented from leaking from the outside of the measurement unit 10 to the inside thereof, and the evaluation sample installed in the sample installation unit 11 is kept in the dark. Such standby is similarly performed for the standard sample (step S13b, second excitation step).

Subsequently, by using the light source 12, the second excitation light is irradiated under the second irradiation condition with respect to the evaluation sample that is kept in a predetermined light condition (step S14a, second excitation step). The second irradiation condition includes a condition such that the integrated light energy value is smaller than that of the first irradiation condition. The light source control unit 31 outputs a control signal to the light source 12 so that the excitation light emitted from the light source 12 satisfies the second irradiation condition. Here, the energy integrated value refers to the light energy integrated value for the photosynthetic sample, and the photon flux density (μmol · m ⁻² · s ⁻¹ ), the photosynthetic effective radiation absorption rate (%) of the photosynthetic sample, and the irradiation. Expressed as the product of time (seconds). Such irradiation of the second excitation light is similarly performed on the standard sample (step S14b, second excitation step).

  Subsequently, the amount of delayed light emission generated from the evaluation sample irradiated with the second excitation light is measured (step S15a, measurement step). Delayed light emission generated from the evaluation sample passes through the filter 13 and is collected by the condensing optical system 14. The condensed delayed light emission is sent to the detection unit 16, and the light emission amount of the delayed light emission is measured by the detection unit 16. Such measurement is performed at predetermined time intervals (for example, every 0.1 second) based on a control signal output from the detection control unit 32 to the light amount calculation unit 16b. The measured light emission amount of delayed light emission is associated with the measurement time or measurement time, output from the detection unit 16 to the analysis unit 20, and recorded in the recording unit 21. Such measurement is similarly performed on the standard sample (step S15b, measurement step).

  The above-described processing from the irradiation of the first excitation light to the measurement of the light emission amount of delayed light emission is repeated a plurality of times while changing the second irradiation condition (step S16a; YES, step S16b; YES). Here, the second irradiation condition is, for example, the second excitation light irradiation start time, in other words, the time from the end of the first excitation light irradiation to the start of the second excitation light irradiation (hereinafter referred to as the second excitation light irradiation start time). "Waiting time"). Such a second irradiation condition is set by the light source control unit 31. When the second irradiation condition is changed, the oxidation-reduction state changes. Therefore, by adopting such a measurement method, it is possible to measure the light emission amount of delayed luminescence under conditions where the oxidation-reduction state is different.

  Note that the number of times of repeating the process from the irradiation of the first excitation light to the measurement of the light emission amount of delayed light emission is not limited, and an arbitrary number of times can be set. It is also possible to prepare a plurality of comparative samples having the same conditions, and measure the delayed luminescence by changing the second irradiation condition for each. In this case, the sample is not affected by the previous measurement, and more accurate measurement can be performed. Furthermore, the time-lapse data of the light emission amount of delayed light emission measured by changing the second irradiation condition or the evaluation value based on the data may be stored in advance in the storage unit. In this case, the delayed luminescence measurement need not be repeated.

  After repeating the process from the irradiation of the first excitation light to the measurement of the light emission amount of delayed luminescence a plurality of times (step S16a; NO, step S16b; NO), the photosynthetic electron transfer of the photosynthetic sample and others are obtained from the obtained measurement results. An analysis value that reflects the metabolism of is calculated (step S17a and step S17b, evaluation step). This calculation is performed for each of the evaluation sample and the standard sample. Specifically, the calculation unit 22 calculates the analysis value by applying the measurement result read from the recording unit 21 to a predetermined calculation program. As the analysis value, for example, an integrated value of the amount of delayed luminescence accumulated every one or more measurement time zones, a measurement time (measurement time) at which a specific change appears in the process of decaying the amount of delayed luminescence, and A coefficient obtained by fitting the time-dependent change in the light emission amount of delayed light emission as the sum of the functions can be mentioned.

  In addition, it is preferable to irradiate the sample with only the second excitation light to measure the light emission amount of delayed light emission and use the light emission amount when calculating the analysis value. In this case, since the amount of delayed luminescence when the sample is not saturated is reflected in the analysis value, the photosynthetic function of the photosynthetic sample included in the evaluation sample can be more appropriately evaluated. When calculating the analysis value, all of the measurement results may be used, or only a part of the measurement results may be used.

  In particular, an integrated value obtained by integrating the measured light emission amount of delayed light emission for each of one or more measurement time zones is preferably used as the analysis value. Thereby, it becomes possible to easily and appropriately extract the redox state of the photosynthetic sample in a certain measurement time zone, which appears in the time-dependent change in the amount of delayed luminescence. In addition, by providing multiple measurement time zones and calculating the integrated value for each measurement time zone, it is possible to properly grasp the change over time in the amount of delayed luminescence, so the oxidation-reduction state of the photosynthetic sample can be more appropriately determined. Can be evaluated.

  It is also preferable to use the coefficient values of a plurality of functions derived so that the time-dependent change in the measured amount of delayed light emission is fitted as a sum of a plurality of preset functions as analysis values. By using such a mathematical method, an evaluation value can be derived objectively. In addition, since the time-dependent change in the amount of delayed light emission is fitted to the sum of a plurality of functions, evaluation based on the mechanism of delayed light emission is possible.

  Note that the above-described processing from preparation to analysis may be performed simultaneously on the evaluation sample and the standard sample, or may be performed at different times. Further, when standard data to be described later is obtained, measurement of a standard sample is not necessarily performed.

  Subsequently, an evaluation value is calculated (derived) based on the analysis value of the evaluation sample and the analysis value of the standard sample (step S18, evaluation step). The calculation of the evaluation value is executed in the calculation unit 22 as with the calculation of the analysis value. The method for calculating the evaluation value is not limited to one. For example, for each of the evaluation sample and the standard sample, the evaluation value is calculated by comparing the analysis values under different oxidation-reduction conditions or by comparing the analysis value of the evaluation sample with the analysis value of the standard sample. Etc. Further, the evaluation values calculated as described above may be compared with each other to further calculate the evaluation value. Furthermore, a method of using the analysis value as an evaluation value as it is, a method of using the result of adding / subtracting / dividing / dividing the analysis value as an evaluation value, and a method of substituting the analysis value into a predetermined function and using the result of calculation as an evaluation value may be adopted. Good. The type and number of evaluation values calculated are not limited.

  Finally, the state of the photosynthetic sample is evaluated based on the calculated evaluation value (step S19, evaluation step). Specifically, the evaluation is performed by comparing the evaluation value obtained by calculation with the standard data. As standard data, the evaluation value obtained based on the measurement data of a standard sample is mentioned, for example. Further, the evaluation value may be evaluated by calculating a reference value from the evaluation value of the standard sample. Examples of the reference value include a threshold value and a range. The evaluation result about the state of the photosynthetic sample obtained here is displayed on the display unit 23 and presented to the evaluator. In addition, the presentation method to an evaluator is not restricted to this, For example, you may show the evaluation value to an evaluator at the time of calculating an evaluation value.

  According to such a photosynthesis sample evaluation method, the amount of delayed luminescence can be measured for each different waiting time, so that a more detailed evaluation value can be derived, and as a result, included in the evaluation sample. It is possible to appropriately evaluate the redox state of the photosynthetic sample.

(Measurement example of delayed luminescence)
Next, measurement of delayed luminescence performed while changing the oxidation-reduction state will be described with reference to measurement examples.

Here, green algae (Pseudokirchneriella subcapitata) is used as a photosynthesis sample. The green algae uses a standard C (75) medium (ph7.5), and is swirled (120 rpm) while irradiating with white light of 50 μmol · m ⁻² · s ^{−1 in} an environment at a temperature of 25 ° C. ± 1 ° C. It was grown in The measurement sample was prepared by using C (75) medium as a diluent and adjusting to 685 nm absorbance OD685 = 0.05, and then irradiating with white light of 50 μmol · m ⁻² · s ^{−1 in} an environment of air temperature 25 ° C. ± 1 ° C. While the cell suspension was cultured for 1 hour, 2.5 mL. And this measurement sample is left still in the dark for 60 seconds before irradiating excitation light and measuring delayed light emission. This is to make the photosynthesis electron transfer in the photosynthetic sample smoothly proceed (to keep the redox state of the photosynthetic sample in a constant state). Normally, the measurement is allowed to stand in the dark for 60 seconds, so that PSII, pQpool, and PSI are sufficiently oxidized.

  In the measurement of delayed light emission, delayed light emission emitted from a measurement sample after irradiation of excitation light by the light source 12 is detected by the detection unit 16, and the detected signals are integrated every 0.1 second to correlate with the light emission amount of the delayed light emission. This is performed by a method of outputting as a value to be counted. This measurement is continuously performed, for example, for 50 seconds after the excitation light is turned off. In general, the amount of delayed emission after excitation light extinguishes attenuates as the elapsed time (time after excitation) elapses from the end of excitation light irradiation.

  First, the relationship between the standby time and the change over time of delayed light emission will be described with reference to FIG. FIG. 5 is a graph showing the relationship between the standby time and the change over time in delayed light emission. The horizontal axis of this graph represents the post-excitation time (elapsed time from the end of irradiation with the second excitation light) (seconds), and the vertical axis represents delayed emission intensity (counts). Since the intensity of delayed emission correlates with the amount of delayed emission, the evaluation based on the intensity of delayed emission in the following is equivalent to the evaluation based on the intensity of delayed emission. is there. In the following, the graph of the relationship between the time after excitation and the intensity of delayed emission is referred to as an attenuation curve.

In obtaining the relationship between the waiting time and the time-dependent change in delayed emission, first, the measurement sample is irradiated with white light of 500 μmol · m ⁻² · s ⁻¹ for 10 seconds as the first excitation light that saturates photosynthetic electron transfer. (First irradiation condition). Thereafter, a step (hereinafter referred to as “standby”) in which the measurement sample is not irradiated with light is provided, and then, 50 μmol · m ⁻² · s ⁻¹ of red light is irradiated as second excitation light for 2 seconds. This second excitation light is capable of generating delayed light emission with almost no change in the redox state of the photosynthetic sample. Such processing is repeated while changing the standby time to 0, 2, 10, 30, and 60 seconds. In addition to these, measurement is also performed when only the second excitation light is irradiated without performing the first excitation light irradiation and standby, that is, when the photosynthetic electron transfer is not saturated.

  The graph shown in FIG. 5 represents the relationship between the standby time and the change over time in delayed light emission in each of these cases. As shown in this graph, by combining the irradiation of the first excitation light, the standby and the irradiation of the second excitation light, a state in which the redox state of the photosynthetic sample is different is created in stages, and the redox state is The reflected delayed luminescence can be measured. In the case of “no saturation” in which the first excitation light is not irradiated, photosynthetic electron transfer proceeds most smoothly. On the other hand, when the second excitation light is irradiated immediately after the first excitation light is irradiated (when the standby time is 0 second), the photosynthetic electron transfer is closest to saturation. As the standby time becomes longer, saturation of photosynthetic electron transfer is eliminated (the photosynthetic electron transfer system of the photosynthetic sample is restored).

  Looking at the decay curve when the waiting time is 0 seconds shown in FIG. 5, the amount of light emission from 0.1 seconds to 0.5 seconds after excitation is higher than that without saturation, and conversely after excitation. From about 0.5 seconds to about 10 seconds, the amount of light emission is reduced compared to the case without saturation. In addition, the amount of light emission is slightly increased from about 10 seconds to 50 seconds after excitation as compared with the case without saturation. The decrease in the amount of light emission from around 10 seconds to 50 seconds after excitation compared to the case without saturation is slightly reduced when the standby time is 2 seconds. Such a change in the attenuation curve approaches the case of no saturation as the standby time increases, and there is almost no difference between the attenuation curve when the standby time is 60 seconds and the attenuation curve without saturation. . From this, it is considered that the saturation of the photosynthetic electron transfer system by the first excitation light is eliminated during the standby time of 60 seconds.

  The reason why the above measurement result is considered to represent a change in the redox state of the photosynthetic sample will be described. As described above, the redox state of the photosynthetic sample is determined by the light conditions of the excitation light to be irradiated, more specifically, the amount of light energy of the excitation light. The factors that determine the amount of light energy include, for example, the amount of irradiation light and the irradiation time. Therefore, for the sake of explanation, first, the relationship between the irradiation light amount and irradiation time and the temporal change of delayed light emission will be shown.

FIG. 6 is a graph showing the relationship between the irradiation light amount of excitation light and the temporal change of delayed light emission. The horizontal axis and vertical axis of this graph represent the same as in FIG. In obtaining the relationship between the irradiation light amount of the excitation light and the temporal change of the delayed light emission, the above measurement is performed while changing the irradiation light amount of the first excitation light to 50, 100, 200 and 500 μmol · m ⁻² · s ^−1. (The standby time was set to 0 second). In addition, when the irradiation light quantity of 1st excitation light is 50 micromol * m ^<-2 > * s ^{< -1} >, saturation of a photosynthetic electron transmission system does not generate | occur | produce. Referring to FIG. 6, as the amount of irradiation light of the first excitation light decreases, the attenuation curve approaches the case of no saturation (when the amount of irradiation light of the excitation light is 50 μmol · m ⁻² · s ⁻¹ ).

  FIG. 7 is a graph showing the relationship between the irradiation time of the excitation light and the change with time of delayed light emission. The horizontal axis and vertical axis of this graph represent the same as in FIG. In obtaining the relationship between the irradiation time of the excitation light and the time-dependent change of the delayed light emission, the above measurement (the standby time was set to 0 second) while changing the irradiation time of the first excitation light to 5, 10 and 30 seconds. ) Was repeated. Referring to FIG. 7, as the irradiation time of the first excitation light decreases, the attenuation curve approaches the case of no saturation (when excitation light is not irradiated).

  As shown in FIGS. 6 and 7, the change in the attenuation curve depends on the irradiation light amount and irradiation time of the first excitation light. That is, as shown in FIG. 5, the increase or decrease in the amount of light emission in a specific measurement time zone caused by the length of the waiting time directly or indirectly reflects the saturation and recovery of the photosynthetic electron transport system due to light irradiation. Conceivable.

  This point will be described in detail with reference to FIGS. 8 to 10 in consideration of the movement of molecules involved in photosynthetic electron transfer while comparing the redox state and the occurrence of delayed luminescence. FIG. 8 is a schematic diagram showing a comparison between a redox state and delayed light emission in a state without saturation. FIG. 9 is a schematic diagram showing a comparison between the oxidation-reduction state and delayed light emission when no standby time is provided after irradiation of the first excitation light (standby time 0 seconds). FIG. 10 is a schematic diagram showing a comparison between the redox state and delayed light emission when a standby time of 2 seconds is provided after the first excitation light irradiation.

  When there is no saturation, the measurement sample is irradiated with excitation light having the same light intensity as that during the cultivation of the photosynthetic sample (green algae). In this case, as shown in FIG. 8, no saturation occurs in the process of transferring electrons from PSII to PSI via pQpool, and oxidized plastoquinone and reduced plastoquinone exist in pQpool in a well-balanced manner. Delayed luminescence occurs because the electrons in the three major electron storage moieties (PSII, pQpool, PSI) that affect delayed luminescence eventually chemically re-excite P680 by the reverse reaction. Note that the measured delayed emission is the sum of the delayed emissions generated from each of these three electron storage portions.

When the standby time is 0 second, the delayed emission is measured after the second excitation light is irradiated immediately after the first excitation light is irradiated. Therefore, it can be said that the photosynthetic electron transfer system is in the most saturated state. In this case, as shown in FIG. 9, in pQpool, the amount of reduced plastoquinone produced by irradiation with strong light increases, while the amount of oxidized plastoquinone decreases. In such a situation, reoxidation of reduced plastoquinone by cytb ₆ f becomes rate limiting, and there is a shortage of oxidized plastoquinone that can accept electrons from PSII. For this reason, extraction of electron transmission from the inside of PSII is reduced, and accumulation of electrons inside PSII is increased. As a result, delayed emission reflecting the electron accumulation inside PSII increases. As shown in FIG. 5, the increase in delayed light emission reflecting the electron accumulation inside PSII is as follows. As shown in FIG. 5, the light emission amount between 0.1 seconds and 0.5 seconds after excitation in the decay curve when the standby time is 0 seconds. Appears as an increase in

  It should be noted that the delayed luminescence in the case where time has not passed so much after excitation reflects the electrons of PSII, Kretsch, G. and Gerhardt, V., “Numerical analysis of delayed fluorescence kinetics of algae”, Archiv furHydrobiologie-Beiheft Ergebnisse der Limnologie, 1987, 29, p. 47-54 (hereinafter referred to as “Reference 1”), and Schmidt, W. and Senger, H., “Long-term delayed luminescence in Scenedesmus obliquus I. Spectral and kinetics properties ”, Biochemica et Biophysica Acta, 1987, 890, p. 15-22 (hereinafter referred to as“ Reference 2 ”).

As the path for backflow electrons to P680 of PSII from PQpool, a path through the oxidized plastoquinone bonded to _{Q B} site of PSII, equilibrium of reduced plastoquinone moved to PQpool dissociates from _{Q B} site There are two possible routes that reflect this. Two pathways they expected, albeit at the reaction via the Q _B site inside PSII, because it is delayed luminescence that reflects the redox state of PQpool, treated as the delayed luminescence that reflects the electron accumulation PQpool. In a situation where photosynthetic electron transfer is saturated and a large amount of reduced plastoquinone is present, delayed luminescence reflecting oxidized plastoquinone decreases, while delayed luminescence reflecting reduced plastoquinone increases.

As shown in the decay curve of the waiting time of 0 seconds in FIG. 5, the decrease in delayed luminescence reflecting the decrease in oxidized plastoquinone appears as a decrease in luminescence from about 0.5 seconds to about 10 seconds after excitation. it is conceivable that. Moreover, it is considered that the increase in the amount of luminescence accompanying the increase in reduced plastoquinone appears as an increase in the amount of luminescence from about 10 seconds to 50 seconds after excitation. The decrease in the luminescence amount that reflects the reduced form plastoquinone appears earlier in time than the increase of the luminescence amount that reflects the reduced form plastoquinones the better of oxidized plastoquinone bonded to Q _B sites within PSII, This is thought to be because the reaction proceeds in a shorter time than reduced plastoquinone that moves in the PSII and thylakoid membrane to create an equilibrium state. It is described in Reference Document 1 that delayed light emission reflecting pQpool emits light later than delayed light emission reflecting PSII.

Furthermore, as a path for the electrons accumulated after PSI to flow back to P680, a path reflecting the reverse reaction of the equilibrium state through the oxidation reaction of reduced plastoquinone from P700 to cytb ₆ f, ferredoxin (Fd of PSI) ) Through the electron transfer to the oxidized plastoquinone in cytb ₆ f to generate reduced plastoquinone. In this case, in either route, PSI electrons flow back to P680 via reduced plastoquinone. As indicated by the decay curve with a waiting time of 0 seconds in FIG. 5, it is considered that the increase in delayed light emission from PSI via reduced plastoquinone appears as an increase in light emission from about 10 seconds to 50 seconds after excitation. It is done. It is described in Reference Document 1 and Reference Document 2 that delayed emission reflecting PSI emits light within a range of several tens of seconds after excitation.

  That is, the increase in the amount of light emission from around 10 seconds to 50 seconds after excitation is due to both the reduction of pQpool accompanying light irradiation and the reduction of pQpool reflecting the cyclic electron transfer from PSI and the equilibrium state with P700. Reflects reduced plastoquinone. In this specification, the delayed luminescence measured between 10 seconds and 50 seconds after the excitation is mainly treated as reflecting the equilibrium state between the cyclic electron transfer from the PSI and P700.

  When the waiting time is 2 seconds, after the first excitation light is irradiated, the waiting time is provided for 2 seconds, and then the second excitation light is irradiated to measure delayed emission. Therefore, it can be said that this measurement result is a point in time when the saturation of the photosynthetic electron transfer system is slightly recovered as compared with the case where the standby time is set to 0 seconds. In this case, as shown in FIG. 10, since oxidized plastoquinone is generated during the waiting time, electrons can be extracted from PSII, and from 0.1 seconds to 0 after excitation observed at the waiting time of 0 seconds. No increase in the amount of light emitted around 5 seconds is observed. In addition, since the oxidized plastoquinone can move to the QB site, the degree of decrease in the amount of light emission from about 0.5 seconds to about 10 seconds after excitation is slightly reduced.

  From this, the degree of increase in delayed luminescence from about 10 seconds to 50 seconds after excitation reflecting the reduced plastoquinone should be reduced as well. However, when the standby time is provided for 2 seconds, the delayed light emission from about 10 seconds to 50 seconds after excitation is increased as compared with the case where the standby time is 0 seconds. This is considered to be due to the backflow of electrons after PSI. That is, it can be said that the delayed light emission from about 10 seconds to 50 seconds after excitation, which is increased by increasing the standby time, mainly reflects electrons after PSI.

  As described above, the electrons in PSII, pQpool, and PSI accompanying the saturation and recovery of photosynthetic electron transfer based on the decay curves of delayed luminescence obtained from the measurement samples having different redox states as shown in FIG. Accumulation can be evaluated. For example, among the time after excitation, the time zone during which the increase or decrease in the amount of light emission is characteristically observed is 0.1 to 0.5 seconds after excitation, 0.6 to 10 seconds after excitation, and 10.1 to 50 after excitation. Focusing on the change in the amount of light emission in each time zone of seconds, such an electron accumulation can be clearly evaluated.

  As described above, by deriving an evaluation value for the evaluation sample based on the light emission amount of each delayed luminescence obtained while changing the irradiation start time of the second excitation light, the redox of the photosynthetic sample included in the evaluation sample is obtained. The state can be evaluated appropriately. In addition, the amount of delayed emission is measured after the second excitation light is irradiated so that the integrated energy value is lower than that of the first excitation light. Therefore, it is possible to prevent the delayed emission from being attenuated without hindering the change in the redox state due to the irradiation of the first excitation light by the irradiation of the second excitation light. As a result, the amount of delayed light emission can be measured with higher accuracy.

(Evaluation of sample by integrated value)
Evaluation of electron accumulation in PSII, pQpool, and PSI in this way is useful for evaluating samples including photosynthetic samples. Specifically, it is useful for evaluating environmental factors present in an evaluation sample including a photosynthetic sample. More specifically, for the evaluation of photosynthetic electron transfer efficiency of photosynthetic samples, evaluation of chemical substances that inhibit the photosynthetic reaction, plant growth diagnosis, influence of harmful substances on plants, functional analysis of pesticides and photosynthesis research Useful. Therefore, a method for evaluating a sample based on the integrated value of the measured amount of delayed luminescence (intensity of delayed luminescence) will be described.

  In the following, integration is performed for an integration range (measurement time zone) of 0.1 to 0.5 seconds after excitation, an integration range of 0.6 to 10 seconds after excitation, and an integration range of 10.1 to 50 seconds after excitation. A case where the value is calculated will be described. The setting of the integration range is not limited to this. The number of integration ranges may be any number as long as it is 1 or more. In addition, when a plurality of integration ranges are provided, whether or not the time widths of the integration ranges are equal is arbitrary. Furthermore, as in the present embodiment, the integrated ranges may not only be separated from each other by a predetermined time, but may be provided so that the integrated ranges are in contact with each other (for example, an integrated time of 0.1 to 0.5 seconds after excitation). Range and an integrated range of 0.5 to 10 seconds after excitation), and may be provided so that the integrated ranges overlap in a predetermined time zone (for example, 0.1 to 0.5 seconds after excitation) An integration range and an integration range of 0.4 to 10 seconds after excitation are provided).

  FIG. 11 is a graph showing the results of calculating integrated values for each of the three integrated ranges for the delayed emission intensity decay curve shown in FIG. In each graph of FIG. 11, the horizontal axis represents the measurement conditions, and the vertical axis represents the integrated value (counts) of delayed emission intensity. In the following, the integrated value in the integrated range of 0.1 to 0.5 seconds after excitation, the integrated value in the integrated range of 0.6 to 10 seconds after excitation, and the integrated range of 10.1 to 50 seconds after excitation The integrated values at are referred to as integrated value 1, integrated value 2, and integrated value 3, respectively. FIG. 11A, FIG. 11B, and FIG. 11C are graphs for integrated value 1, integrated value 2, and integrated value 3, respectively.

  First, the examination is based on the integrated value 1. As shown in FIG. 11A, the integrated value 1 when the standby time is 0 second is higher than that when there is no saturation. On the other hand, the difference between the integrated value 1 when the waiting time is 2 seconds and that without saturation is not large. The change in the case where the waiting time is 0 second is that reduced plastoquinone is excessively formed by irradiation with the first excitation light, and the oxidized plastoquinone that binds to the QB site in PSII and accepts electrons from inside PSII is This is a result of increased electron accumulation in PSII due to lack. On the other hand, when the standby time is set to 2 seconds or more, oxidized plastoquinone is generated during the standby time, and electrons in PSII are taken out. That is, the integrated value 1 reflects the accumulation of electrons inside the PSII. When the integrated value 1 is high, it indicates that there are a lot of electrons stored in the PSII and that the reduced state is present.

  This is examined from the viewpoint of the change due to the difference in the redox state of the photosynthetic sample. Under normal conditions of photosynthetic electron transfer, pQpool is reduced immediately after irradiation of excitation light that saturates electron transfer (for example, irradiation of the first excitation light), and oxidized plastoquinone that can bind to the QB site is present. Run short. For this reason, the integrated value 1 temporarily increases, but is resolved by a condition (for example, waiting for a predetermined time) in which the saturated state is recovered. Therefore, when the increment of the accumulated value 1 of the waiting time 0 seconds (closest to saturation) compared to the accumulated value 1 in the case of no saturation represents the electrons accumulated in the PSII due to the saturation of the electron transfer. I can say that. That is, the process of decreasing the increment under conditions for recovering the saturation (for example, waiting for a predetermined time) reflects the extraction of electrons from PSII by oxidized plastoquinone. Therefore, by paying attention to the difference between the integrated values 1 in which the redox states of the photosynthetic samples are different from each other, it is possible to evaluate the effects of environmental factors (for example, harmful substances) on the accumulation of electrons in PSII and the extraction of electrons. .

  Next, consideration is made based on the integrated value 2. As shown in FIG. 11B, the integrated value 2 is the smallest value in the case of the standby time of 0 seconds compared to the value in the case of no saturation. The difference decreases as the standby time becomes longer, and in the case of the standby time of 60 seconds, the integrated value 2 is restored to the same level as in the case of no saturation. That is, the integrated value 2 reflects oxidized plastoquinone that can bind to the QB site of PSII and accept electrons. When the waiting time is 0 second, excessively reduced plastoquinone is formed by irradiation with the first excitation light, and there is a shortage of oxidized plastoquinone that binds to the QB site in PSII and can accept electrons from inside PSII. To do. For this reason, delayed luminescence reflecting the electron accumulation of plastoquinone bound to the QB site is reduced. However, as the waiting time increases, the production of oxidized plastoquinone proceeds, and the amount of delayed luminescence via the QB site is recovered.

That is, the difference of the integrated value 2 of the waiting time 0 seconds (the state closest to saturation) compared to the integrated value 2 in the case of no saturation represents the reduction of pQpool due to intense light irradiation. In addition, the process in which the difference decreases under the condition that saturation is restored (for example, waiting for a predetermined time) represents the process in which reduced plastoquinone is reoxidized by cytb ₆ f or the like. Therefore, by paying attention to the difference between the integrated values 2 having different redox states, it is possible to evaluate the effect of environmental factors on the reduction state of pQpool and the reoxidation process.

  Next, the examination is based on the integrated value 3. As shown in FIG. 11C, the integrated value 3 increases most when compared with the value when there is no saturation when the standby time is 2 seconds, and is equal to when there is no saturation when the standby time is 60 seconds. It has decreased to the extent that it becomes. It can be said that the integrated value 3 when the standby time is provided for 2 seconds or more corresponds to reduced plastoquinone mainly reflecting the accumulation of electrons after PSI. That is, the increment of the accumulated value 3 in each condition of the standby time of 0 to 60 seconds compared with the accumulated value 3 in the case of no saturation reaches PSII from the PSI onward by cyclic electron transfer or reverse reaction via the P700. Represents the flow of electrons. Therefore, by paying attention to the difference between the integrated values 3 having different oxidation-reduction states, it is possible to evaluate the effects of environmental factors on the accumulation of electrons after PSI and the cyclic electron transfer.

  By using such an integrated value, it is possible to more appropriately extract the redox state of the photosynthetic sample that appears in the temporal change in the amount of delayed luminescence. As a result, the oxidation-reduction state of the photosynthetic sample included in the evaluation sample can be appropriately evaluated. In addition, as described above, when a plurality of integrated values are calculated, it is possible to grasp the change over time in the amount of delayed luminescence, so that the redox state is appropriately evaluated by using this change over time. be able to.

  From the above, as a method of evaluating the saturation and recovery of the photosynthetic electron transport system, a plurality of states having mutually different redox states are prepared, delayed luminescence is measured for each of these states, the PSII interior, the plastoquinone pool, and the electrons after PSI It can be seen that it is effective to use an index (for example, the integrated values 1, 2, and 3) representing delayed light emission that reflects the accumulation.

(Evaluation of environmental factors)
Next, a method for evaluating the influence of environmental factors on the photosynthetic sample by evaluating the saturation and recovery of the photosynthetic electron transport system will be described. At that time, as an evaluation method, an evaluation method using the integrated values 1, 2, and 3 is used.

Here, a standard sample (Cont) and an evaluation sample are prepared. The green algae similar to those described above are used as the photosynthetic sample contained in each sample. Specifically, C (75) medium is used as a diluent, and the absorbance at 685 nm is adjusted to OD685 = 0.05, and white light of 50 μmol · m ⁻² · s ⁻¹ is irradiated in an environment at a temperature of 25 ° C. ± 1 ° C. Then, 2.5 mL of the cell suspension cultured for 1 hour is used as a standard sample. On the other hand, after preparing the evaluation sample in the same procedure as that of the standard sample, when culturing for 1 hour while irradiating white light of 50 μmol · m ⁻² · s ^{−1 in} an environment at an air temperature of 25 ° C. ± 1 ° C., As an environmental factor, it is obtained by adding ultraviolet rays of 365 nm with an intensity of 100 μW / cm ² (UV irradiation). The amount of delayed luminescence is measured by leaving the standard sample and the evaluation sample in the dark for 60 seconds in advance to remove the influence of the environment before measurement on the photosynthetic electron transport system, and making the photosynthetic electron transport system constant oxidation-reduction. Went after unifying to the state.

  FIG. 12 is a graph showing the relationship between the standby time and the time-dependent change in the amount of delayed luminescence in the standard sample. FIG. 13 is a graph showing the relationship between the standby time and the time-dependent change in delayed light emission in the evaluation sample. Each graph shows changes over time in the amount of delayed light emission in each case of no saturation, standby time 0 seconds, standby time 10 seconds, and standby time 60 seconds. Comparing FIG. 12 with FIG. 13, it can be seen that the shape of the attenuation curve is characteristically changed by UV irradiation.

  Hereinafter, the influence of UV irradiation will be described in detail. FIG. 14 is a graph showing integrated values 1, 2, and 3 for each sample calculated based on the light emission amount of delayed light emission shown in FIGS. 12 and 13, and FIGS. 14 (a) and 14 (b). FIG. 14C is for the integrated value 1, integrated value 2 and integrated value 3, respectively.

  First, looking at the standard sample, the integrated value 1 reflecting the electrons inside PSII shows the maximum value when the standby time is 0 seconds, and is the same as when there is no saturation when the standby time is 10 seconds and 60 seconds. The value of is shown. As described above, immediately after the first excitation light irradiation (when the waiting time is 0 second), there are many reduced plastoquinones in pQpool and there are few oxidized plastquinones that can extract electrons from PSII. This is because the accumulation of electrons inside PSII increases. On the other hand, in the case of the waiting time of 10 seconds and 60 seconds, since the oxidized plastoquinone is generated during the waiting time, the photosynthetic electron transfer system is not saturated in which the PSII electron transfer reaction proceeds smoothly. Recovery to the same level as in the case of.

On the other hand, the integrated value 2 reflecting the electrons of pQpool shows a minimum value at a waiting time of 0 seconds, and gradually increases as the waiting time becomes longer and approaches a value without saturation. This is because, as described above, when it immediately first excitation light irradiation (for standby time 0 sec), reduced plastoquinone many exist in PQpool, the Q _B site is plastoquinone binding site within the PSII This is because there is a shortage of oxidized plastoquinone that can be bound. On the other hand, when the standby time is provided for a predetermined time, oxidized plastoquinone is generated during the standby time, so that the photosynthetic electron transfer system recovers to the same level as in the case of no saturation.

  Further, the integrated value 3 reflecting the electrons after the PSI takes almost the same value when there is no saturation, the standby time is 0 second, and the standby time is 60 seconds, and the value increases only when the standby time is 10 seconds. This is due to delayed luminescence due to cyclic electron transfer reaction in which electrons stored in ferredoxin and NADPH after PSI are transferred to oxidized plastoquinone via ferredoxin to produce reduced plastoquinone, and reverse reaction via P700. It reflects. The delayed luminescence reflected in the integrated value 3 passes through many reaction paths compared to the delayed luminescence reflected in the integrated value 1 or the integrated value 2, so that the standby time is 10 seconds than the standby time of 0 seconds. The case takes a higher value. This integrated value 3 recovers to the same level as in the case of no saturation when the standby time is 60 seconds.

  Next, looking at the evaluation sample subjected to UV irradiation, the integrated value 1 reflecting the electrons in the PSII does not increase when the standby time is 0 seconds, and there is a large change even when the standby times are 10 seconds and 60 seconds. I can't see it. This suggests that some change has occurred in PSII due to ultraviolet irradiation.

  On the other hand, the integrated value 2 reflecting the electrons of pQpool is slightly decreased compared with that of the standard sample, but shows a minimum value at a waiting time of 0 seconds, and gradually becomes not saturated as the waiting time becomes longer. The trend of getting closer to the value does not change. This suggests that UV irradiation does not significantly affect the action of pQpool.

  Further, the integrated value 3 reflecting the electrons after the PSI is almost the same value in the case of no saturation and in the case of the standby time of 60 seconds, but in addition to the case of the standby time of 10 seconds, the standby time of 0 seconds. Even if the value is increasing. This suggests that the irradiation with ultraviolet rays increased the cyclic electron transfer reaction that is transmitted to oxidized plastoquinone via PSI to produce reduced plastoquinone and the reverse reaction via P700.

(Evaluation value calculation method)
Next, an evaluation value calculation (derivation) method in the evaluation step will be described. In this explanation, the second irradiation condition is the above-described standby time (the standby time is 0 second, the standby time is 10 seconds, and the standby time is 60 seconds), and the evaluation value is based on the integrated values 1, 2, and 3. Assuming Further, it is assumed that the integrated values 1, 2 and 3 are calculated in the same manner even in the case of no saturation.

  First, a method for calculating an evaluation value for evaluating the electron accumulation state of PSII of the photosynthetic sample will be described. As one of such evaluation values, for example, there is a change (eII) in PSII electron accumulation indicating how the PSII electron accumulation has changed. This value eII can be obtained by (integrated value 1 of evaluation sample) / (integrated value 1 of standard sample) × 100. The PSII electron accumulation change (eII) can be calculated for the following conditions: no saturation, waiting time 0 seconds, waiting time 10 seconds, and waiting time 60 seconds.

  A change in PSII electron accumulation (eII) of more than 100 indicates an increase in PSII electron accumulation. For example, if this eII is calculated under the condition of no saturation, it is possible to evaluate the change in the electron accumulation of PSII under a standard condition without the influence of strong light (for example, in the case of no saturation).

  As described above, the transient increase in the integrated value 1 at the waiting time of 0 seconds reflects the increase in the PSII electron accumulation due to the rate-determining of the electron extraction from the PSII. The transient increase of the integrated value 1 is restored to the same level as when there is no saturation by providing a standby time during which no light is irradiated. For example, the ability to take out electrons from PSII in a certain standby time can be evaluated by how much the transient increase of the integrated value 1 at the standby time of 0 seconds is reduced by providing the standby time. Therefore, the PSII electron extraction capability (tII) from the standby time of 0 seconds to a certain condition is calculated as an evaluation value. This value tII is ((integrated value 1 when standby time is 0 second) / (integrated value 1 when there is no saturation) − (integrated value 1 when there is no saturation) / (integrated value 1 when there is no saturation)) ) × 100.

  If the electron extraction capability (tII) of the PSII from the standby time of 0 second to a certain standby time is large, it indicates that a lot of PSII transient electron accumulation has been eliminated from the standby time of 0 second to the certain standby time. Conversely, when this value tII is a negative value, it indicates that the PSII electron accumulation has increased for some reason from the waiting time of 0 seconds to a certain waiting time.

  Furthermore, by comparing PSII's electron extraction capability (tII) from a waiting time of 0 seconds to a certain condition for a standard sample that has not been given an environmental factor with that for an evaluation sample that was given an environmental factor, You can see how the electronic take-out has changed. Therefore, as a value indicating this change, a change (vII) in PSII electron extraction from a standby time of 0 second to a certain condition is calculated as an evaluation value. This value vII can be obtained by (tII of evaluation sample) / (tII of standard sample) × 100. If this value vII is 100, it indicates that the ability to extract electrons from PSII has not changed due to environmental factors. On the other hand, when this value vII is small, it indicates that the ability to extract electrons from PSII is reduced, and when it is a negative value, it indicates that electrons are flowing into PSII.

  Moreover, according to the photosynthesis sample evaluation method according to the present embodiment, the redox state of pQpool of the photosynthesis sample can be evaluated. For example, the evaluation value can be derived using the integrated value 2 that reflects the redox state of pQpool.

  In the present embodiment, since the photosynthetic sample is left in the dark for 60 seconds in advance, when there is no saturation, the reduced plastoquinone generated by light irradiation is reoxidized, and pQpool is almost oxidized. Become. In addition, at a waiting time of 0 seconds immediately after irradiation with the first excitation light, almost all pQpool is reduced and there is almost no oxidized plastoquinone. That is, the difference between the integrated value 2 when there is no saturation and the integrated value 2 when the standby time is 0 seconds represents the magnitude of the oxidation-reduction reaction caused by light irradiation. Therefore, the value (sQ) of the oxidation-reduction reaction is calculated as an evaluation value as a value indicating the magnitude. This value sQ can be obtained by (integrated value 2 when there is no saturation) − (integrated value 2 when the standby time is 0 seconds).

  By calculating this value sQ for both the standard sample and the evaluation sample and comparing them, it is possible to know how the size of the redox reaction of the photosynthetic sample has changed due to environmental factors. Therefore, the ratio of the oxidation-reduction reaction magnitude (sQ) for the evaluation sample to the oxidation-reduction reaction magnitude (sQ) for the standard sample is calculated as an evaluation value called change (vQ) due to environmental factors of sQ. This value vQ can be obtained by (sQ of evaluation sample) / (sQ of standard sample) × 100.

As described above, pQpool that is in a substantially reduced state at a waiting time of 0 second is recovered to an unsaturated state because reoxidation reaction by cytb ₆ f proceeds and a oxidized plastoquinone is generated by providing a waiting time. To go. That is, for the integrated value 2 that reflects the redox state of pQpool, by comparing the integrated value 2 at a certain standby time with the integrated value 2 at a standby time of 0 seconds, the amount of oxidized plastoquinone produced at that standby time Can know. The amount of the oxidized plastoquinone decreases the re-oxidation is inhibited by Cytb 6 _f, increases the cyclic electron transfer to reduce the oxidized plastoquinone is inhibited in Cytb 6 _f. Therefore, the amount of oxidized plastoquinone produced (oQ) in a certain waiting time is calculated as an evaluation value. This value oQ can be obtained by (integrated value 2 at a certain standby time) − (integrated value 2 at a standby time of 0 seconds).

  Furthermore, the ratio (% oQ) of oxidized plastoquinone at a certain waiting time to the magnitude of the redox reaction of pQpool can be obtained by (oQ at a certain waiting time) / sQ × 100.

  Moreover, according to the evaluation method of the photosynthetic sample according to the present embodiment, it is possible to evaluate the PSI electron accumulation state of the photosynthetic sample. For example, it is possible to derive an evaluation value using the integrated value 3 that reflects the state of electron accumulation of PSI calculated from the measurement result of delayed light emission.

  Specifically, it is possible to know how the PSI electron accumulation has changed by comparing the integrated value 3 of the standard sample and the integrated value 3 of the evaluation sample in a certain waiting time. Therefore, a change (eI) in PSI electron accumulation is calculated as an evaluation value. The change (eI) in the PSI electron accumulation can be obtained by (Evaluation sample integrated value 3) / (Standard sample integrated value 3) × 100. This value eI can be calculated under the following conditions: no saturation, standby time 0 seconds, standby time 10 seconds, and standby time 60 seconds. That this value eI exceeds 100 indicates that the electron accumulation of PSI is increasing. For example, if the change (eI) in the PSI electron accumulation is calculated under the condition without saturation, the change in the PSI electron accumulation under the standard condition without the influence of strong light can be evaluated.

When the first excitation light as described above is irradiated, the electron accumulation of PSI increases. Some of the electrons of PSI reduce pQpool through a cyclic electron transfer pathway to produce reduced plastoquinone. Many electronic conveyed into reduced plastoquinone results in delayed luminescence in PSII reaction center (P680) via the _{Q B} site and _{Q A} site from PQpool. The integrated value 3 in the case of the standby time of 10 seconds increases temporarily with respect to the integrated value 3 in the case of no saturation. That is, the difference obtained by subtracting the integrated value 3 in the case of no saturation from the integrated value 3 in the case where a certain waiting time is provided represents the cyclic electron transfer from the PSI in the standby time. Therefore, the cyclic electron transfer (cI) from this PSI is calculated as an evaluation value. This value cI can be obtained by (integrated value 3 at a certain standby time) / (integrated value 3 without saturation) × 100.

  As described above, this embodiment can evaluate the change in the redox state. For example, if an environmental factor (for example, ultraviolet rays or chemical substances) is given to the evaluation sample as described above, whether or not the environmental factor is harmful to the photosynthetic sample and what effect the environmental factor has. Can be evaluated. Furthermore, if river water or soil extract water is used when preparing an evaluation sample, environmental factors (harmful or beneficial chemical substances) contained in these waters can be evaluated.

  The usefulness of the evaluation of the change in the redox state shown in the present embodiment is not limited to the above. For example, it can be used for plant growth diagnosis, influence of chemical substances on plants, functional analysis / development of agricultural chemicals, and photosynthesis.

(Evaluation program for photosynthetic samples)
Next, a photosynthesis sample evaluation program (hereinafter simply referred to as “evaluation program”) 50 according to the embodiment will be described with reference to FIGS. 15 and 16. FIG. 15 is a diagram showing the configuration of the evaluation program 50 according to the embodiment together with the recording medium 60. FIG. 16 is a diagram illustrating a hardware configuration of a computer 70 on which the evaluation program 50 illustrated in FIG. 15 operates.

  When the evaluation program 50 cooperates with the computer 70, the computer 70 can operate as the delayed luminescence measuring apparatus 1. Thereby, evaluation of the above-mentioned photosynthetic sample can be realized on the computer 70.

  As shown in FIG. 15, the evaluation program 50 includes a light source control module 51, a detection control module 52, and a calculation module 53. The light source control module 51 corresponds to the light source control unit 31, the detection control module 52 corresponds to the detection control unit 32, and the calculation module 53 corresponds to the calculation unit 22. That is, the computer 70 functions as a first excitation unit and a second excitation unit by the light source control module 51, the computer 70 functions as a measurement unit by the detection control module 52, and the computer 70 functions as an evaluation unit by the calculation module 53.

  As shown in FIG. 16, a computer 70 includes a CPU 71 that executes an operating system, application programs, and the like, a main storage device 72 that includes a ROM and a RAM, an auxiliary storage device 73 that includes a hard disk, and a communication such as a network card. It comprises a control device 74, an output device 76 such as a monitor or printer, and an input device 75 such as a keyboard or mouse. Each module described with reference to FIG. 15 loads predetermined software on the CPU 71 and the main storage device 72 shown in FIG. 16 and operates the communication control device 74 under the control of the CPU 71. And by reading and writing data in the auxiliary storage device 73.

  According to this evaluation program 50, the amount of delayed light emission can be measured for each different second irradiation condition, so that a more detailed evaluation value can be derived. As a result, the redox state of the photosynthetic sample contained in the evaluation sample can be evaluated appropriately and simply.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Green algae was used as a photosynthesis sample. Then, after preparing C (75) medium as a diluent at 685 nm absorbance OD685 = 0.05, it was irradiated with white light of 50 μmol · m ⁻² · s ⁻¹ for 1 hour in an environment of air temperature 25 ° C. ± 1 ° C. 2.5 mL of the cultured cell suspension was used as a standard sample. On the other hand, the evaluation sample is 3- (3,4-dichlorophenyl) -1,1-dimethyl-urea (DCMU) exposed at a concentration of 12 ug / L, 2,5-dibromo-6-isopropyl-3-methyl Those exposed to -1,4-benzoquinone (DBMIB) at a concentration of 322 ug / L and those exposed to Antimycin A (Ant-A) at a concentration of 0.55 mg / L were prepared. These chemical substances are all inhibitors of photosynthetic electron transfer. Hereinafter, the samples exposed to DCMU, DBMIB, and Ant-A are referred to as DCMU exposed samples, DBMIB exposed samples, and Ant-A exposed samples, respectively. Then, the above-described evaluation method was carried out for each of the standard sample and the three types of exposed samples under the following conditions: no saturation, waiting time 0 seconds, waiting time 10 seconds, and waiting time 60 seconds. The redox state of the photosynthetic sample is different under each of these conditions.

  17 to 20 are graphs showing changes over time (decay curves) of the amount of delayed luminescence for the standard sample, the DCMU-exposed sample, the DBMIB-exposed sample, and the Ant-A-exposed sample in the examples, respectively. As shown in FIGS. 17-20, exposure to chemicals changed the shape of the decay curve for each exposed sample.

  Hereinafter, changes in the shape of these attenuation curves will be described in detail using integrated values. For this description, FIGS. 21 to 23 are used. FIG. 21 is a graph comparing the integrated value of the standard sample and the integrated value of the DCMU-exposed sample in the example. FIGS. 21 (a), 21 (b), and 21 (c) show the integrated value 1 respectively. 4 is a graph for integrated value 2 and integrated value 3. FIG. 22 is a graph comparing the integrated value of the standard sample and the integrated value of the DBMIB-exposed sample in the example. FIGS. 22 (a), 22 (b), and 22 (c) show the integrated value 1 respectively. 4 is a graph for integrated value 2 and integrated value 3. FIG. 23 is a graph comparing the integrated value of the standard sample and the integrated value of the Ant-A-exposed sample in the example. FIGS. 23 (a), 23 (b), and 23 (c) are integrated values, respectively. It is a graph about value 1, integrated value 2, and integrated value 3.

  First, the result of the standard sample common to each exposed sample will be described. The integrated value 1 that reflects the electrons inside the PSII showed the maximum value when the standby time was 0 seconds, whereas when the standby time was 10 seconds and the standby time was 60 seconds, it was the same value as no saturation. I took it. As described above, there is a large amount of reduced plastoquinone in pQpool immediately after irradiation with the first excitation light, and there is less oxidized plastoquinone that can extract electrons from PSII, so that the accumulation of electrons inside PSII is reduced. This is because it increases. In the case of the waiting time of 10 seconds and 60 seconds, it can be understood that the oxidized plastoquinone was generated during the waiting time, and the PSII electron transfer reaction was smoothly recovered to the same level as that without saturation. .

  The integrated value 2 reflecting the electrons of pQpool showed a minimum value when the waiting time was 0 second, and gradually approached the value when there was no saturation as the waiting time became longer. As described above, there is a large amount of reduced plastoquinone in pQpool immediately after irradiation with the first excitation light, and there is a shortage of oxidized plastoquinone that can bind to the QB site that is the plastoquinone binding site in PSII. It is because it is doing. Oxidized plastoquinone was produced during the waiting time, and therefore, when the waiting time was 60 seconds, it recovered to the same level as in the case of no saturation.

  The integrated value 3 reflecting the electrons after the PSI becomes almost the same value when there is no saturation, the standby time is 0 second, and the standby time is 60 seconds, and the value increases only when the standby time is 10 seconds. This is because the electrons accumulated in ferredoxin and NADPH after PSI are transferred to oxidized plastoquinone via ferredoxin, resulting in a cyclic electron transfer reaction that produces reduced plastoquinone and a reverse reaction via P700. It is. Since the reaction indicated by the integrated value 3 passes through many reaction paths as compared with the reaction indicated by the integrated value 1 or the integrated value 2, the case where the standby time is 10 seconds is more than the case where the standby time is 0 seconds. The amount of light emission increases. The integrated value 3 recovered to the same level as in the case of no saturation when the standby time was 60 seconds.

Next, the results specific to each exposed sample will be described. First, the result of a DCMU exposure sample is demonstrated. DCMU inhibits the transfer of electrons from inside PSII to pQpool by inhibiting the binding of oxidized plastoquinone to the QB site in PSII. Therefore, the production of reduced plastoquinone is inhibited, and the abundance of oxidized plastoquinone increases in the plastoquinone pool. As a result of the reduced electron accumulation of the plastoquinone pool, the formation of a proton gradient by cytb ₆ f, a decrease in electron supply to photosystem I (PSI) and a decrease in NADPH synthesis occur.

  The integrated value 1 of the DCMU-exposed sample shows a value close to twice the value of the standard sample in each case of no saturation, a standby time of 10 seconds, and a standby time of 60 seconds, and is the closest to saturation in the data regarding the standard sample. The value was higher than in the case of a certain waiting time of 0 seconds. In the standard sample, the integrated value 1 was larger in the waiting time of 0 second (saturated state) than in the case of no saturation, whereas the DCMU-exposed sample had the opposite result. Further, in the DCMU-exposed sample, the value did not decrease even when the saturation was recovered (when the waiting times were 10 seconds and 60 seconds). Thereby, it was found that the electron extraction from the inside of PSII was inhibited regardless of the saturation of electron transfer by intense light irradiation. This is consistent with DCMU inhibiting PSII electron transfer.

  Note that there was no significant difference between the standard sample and the DCMU-exposed sample when the waiting time was 0 seconds because the electron dissipation mechanism such as cyclic electron transfer in PSII immediately after the first excitation light irradiation. This is thought to be due to working.

  The integrated value 2 of the DCMU exposed sample was lower than that of the standard sample in all conditions. However, the minimum value was shown when the waiting time was 0 seconds, and the tendency to gradually approach the value without saturation as the waiting time became longer was the same as the result of the standard sample. This indicates that the electron accumulation in pQpool was reduced, but the process of reoxidizing reduced plastoquinone was not inhibited. This is consistent with DCMU reducing electron transfer to PSII and beyond but not inhibiting other reactions.

  The integrated value 3 of the DCMU exposed sample was lower than that of the standard sample in all conditions. Further, the integrated value 3 of the DCMU-exposed sample was higher than that in the case of no saturation when the waiting times were 0 seconds and 10 seconds. Although there was a difference in increase in the value at a waiting time of 0 seconds, which was not recognized in the standard sample, the tendency was basically the same as that in the standard sample. This indicates that the electron inflow to PSII due to the reverse reaction from PSI onward decreased, but the backflow of electrons from PSI onward was not inhibited. This is consistent with DCMU reducing electron transfer to PSII and beyond but not inhibiting other reactions.

Next, the results of the DBMIB exposed sample will be described. DBMIB binds to the plastoquinone binding site of the cytb ₆ f complex and inhibits oxidation of reduced plastoquinone. Therefore, in pQpool, the abundance of reduced plastoquinone increases and the abundance of oxidized plastoquinone decreases.

  The integrated value 1 of the DBMIB exposed sample was higher than that of the standard sample under all conditions. However, the value increased at a waiting time of 0 seconds as compared to the case without saturation, and recovered to the same extent as when there was no saturation when the waiting times were 10 seconds and 60 seconds, as in the case of the standard sample. Met. This indicates that although the electron accumulation inside PSII increased, the extraction of electrons from PSII itself was not inhibited. This is consistent with DBMIB inhibiting the production of oxidized plastoquinone and reducing the extraction of electrons from PSII.

  When the integrated value 2 of the DBMIB-exposed sample was compared with the integrated value 2 of the standard sample, no significant difference was observed in the case of the standby time of 0 seconds, which was in a state closest to saturation. However, under other conditions (no saturation, waiting time 10 seconds and waiting time 60 seconds), the integrated value 2 of the DBMIB-exposed sample was lower than that of the standard sample. That is, in the DBMIB-exposed sample, the difference between the integrated value 2 in the state closest to saturation (standby time 0 seconds) and the integrated value 2 in the state without saturation was reduced. This is because under the condition of no saturation, pQpool is in a reduced state despite the absence of intense light irradiation, and the amount of oxidized plastoquinone in pQpool is increased. This is consistent with DBMIB inhibiting the oxidation of reduced plastoquinone, but not other electron transport.

  When the integrated value 3 of the DBMIB-exposed sample was compared with the integrated value 3 of the standard sample, there was no significant difference between the two in each case of no saturation, standby time 0 seconds, and standby time 60 seconds. However, when compared with the case where the waiting time was 10 seconds, the value increased relatively large in the standard sample, whereas the increment in the DBMIB-exposed sample was lower than that in the standard sample. This indicates that the flow of electrons from the PSI onwards to pQpool has decreased. This is consistent with DBMIB reducing the abundance of oxidized plastoquinone that serves as an electron acceptor for cyclic electron transfer, one of the electron flow from PSI to plastoquinone.

Next, the results of the Ant-A exposed sample will be described. Ant-A is a chemical substance that inhibits the cyclic electron transfer that produces reduced plastoquinone by transferring electrons to oxidized plastoquinone of cytb ₆ f via PSI ferredoxin (Fd). That is, Ant-A inhibits the reaction of circulating electrons from PSI to pQpool.

  When the integrated value 1 of the Ant-A-exposed sample was compared with the integrated value 1 of the standard sample, no significant difference was observed in each case of no saturation, standby time 0 seconds, and standby time 60 seconds. Further, the same was true in the case where the standby time was 0 second, and the integrated value 1 increased compared to the case where there was no saturation. However, paying attention to the case where the waiting time is 10 seconds, the integrated value 1 decreased to the same extent as in the case of no saturation in the standard sample, whereas in the Ant-A-exposed sample, it increased as in the case of the waiting time 0 second. In the case of a waiting time of 60 seconds, it decreased to the same level as no saturation. Regarding the fact that there was a large amount of electron accumulation inside PSII even in the standby time of 10 seconds, where the electron accumulation inside PSII should have been eliminated, the recovery of saturation was not the case because the electron extraction from PSII was not hindered. Could be late, or electrons could have flowed into PSII from a different path.

In the integrated value 2 of the Ant-A exposed sample, the light emission amount was lower than that of the standard sample under all conditions. However, the minimum value was shown when the waiting time was 0 second, and the tendency to gradually approach the value without saturation as the waiting time became longer was the same as that of the standard sample. This indicates that although oxidized plastoquinone is reduced as compared with the standard sample (pQpool is in a reduced state), the production of oxidized plastoquinone in cytb ₆ f is not inhibited. This is consistent with Ant-A not inhibiting the production of oxidized plastoquinone in cytb ₆ f.

  In the integrated value 3 of the Ant-A exposed sample, the light emission amount was lower than that of the standard sample under all conditions. Moreover, the increase of the integrated value 3 observed in the standard sample at the standby time of 10 seconds was not observed. As described above, since it is considered that cyclic electron transfer is involved in the increase of the integrated value 3 in the waiting time of 10 seconds, this result indicates that the electron accumulation after the PSI decreases and the electrons from the PSI to the pQpool It can be said that this indicates that the flow of water is hindered. The inhibition of electron flow from PSI to pQpool is consistent with Ant-A inhibiting cyclic electron transfer. The decrease in the accumulation of electrons in PSII is also thought to be due to the decrease in electrons flowing from pQpool into PSI again due to the inhibition of cyclic electron transfer. It is also possible that electrons that have lost their destination due to the inhibition of cyclic electron transfer have moved to PSII by some mechanism, and as a result, have caused an increase in integrated value 1 in the above-described waiting time of 10 seconds.

  Next, using FIGS. 24 to 26, values eII, vII, vQ,% oQ, eI obtained by performing the above-described evaluation value calculation method based on the measurement values of the standard sample and each exposed sample in the examples, and The cI will be described. FIG. 24 is a table showing the values eII and vII of each sample without saturation. FIG. 25 is a table showing the values vQ and% oQ for each sample. FIG. 26 is a table showing the values eI and cI of each sample without saturation.

  As shown in FIG. 24, since the value eII of the DCMU-exposed sample without saturation was 188, it can be seen that the electron accumulation in PSII was increased as compared with the case of the standard sample. Further, since the vII of the DCMU-exposed sample was a negative value, it was found that there was no extraction of electrons from PSII, and conversely, electrons were flowing into PSII. This is consistent with the action of DCMU, which inhibits electron extraction from PSII and increases PSII electron accumulation.

Further, as shown in FIG. 25, the DBMIB-exposed sample has a value vQ of 30. This indicates that the redox reaction due to light irradiation is reduced as compared with the standard sample. Further, regarding the value% oQ, the standard sample was 36 at the standby time of 10 seconds and 107 at the standby time of 60 seconds, whereas the DBMIB-exposed sample was 4 at the standby time of 10 seconds and the standby time of 60 seconds. The number was reduced as compared with the standard sample. This indicates that in the DBMIB-exposed sample, the redox reaction of pQpool decreased and the reoxidation of plastoquinone at cytb ₆ f decreased compared to the standard sample. This is consistent with the action of DBMIB, which inhibits plastoquinone reoxidation at cytb ₆ f.

  Further, as shown in FIG. 26, the value eI of the Ant-A-exposed sample without saturation is 89, and the electron accumulation of PSI is not so reduced as compared with the case of the standard sample. However, the value cI of the standard sample with a waiting time of 10 seconds is 38, whereas the value cI of the Ant-A exposed sample with a waiting time of 10 seconds is -12. This shows that in the Ant-A-exposed sample, the electron accumulation of PSI did not change so much, but the inflow of electrons due to cyclic electron transfer from PSI to pQpool decreased. This is consistent with Ant-A's ability to inhibit cyclic electron transfer.

  As described above, from the measurement results using DCMU, DBMIB, and Ant-A, the measurement results are obtained by measuring the delayed luminescence of the photosynthetic samples in different oxidation-reduction states for the standard sample that is not exposed to harmful substances and the exposed sample that is exposed to harmful substances. It was found that the presence or absence of harmful substances and the difference in action on the photosynthetic electron transfer reaction can be evaluated. Such characteristics are useful for evaluating the effects of harmful substances on photosynthetic samples such as algae, represented by chemical ecological risk assessment.

  The present invention has been described in detail based on the embodiments. However, the present invention is not limited to the above embodiment. The present invention can be modified in various ways as described below without departing from the scope of the invention.

  In the above embodiment, the evaluation value is derived based on the integrated value obtained by integrating the measured amount of delayed light emission for each measurement time period. However, the evaluation value deriving method is not limited to this. For example, the coefficient values of the plurality of functions are determined so that the time-lapse data (attenuation curve) of the delayed light emission amount is fitted as a sum of a plurality of preset functions, and the evaluation value is derived based on the coefficient values. May be.

  A method of fitting the time-lapse data of the delayed light emission amount to the time-lapse data function (hereinafter referred to as “fitting method”) will be described in detail. In order to use the fitting method, information of a plurality of functions is stored in advance in the recording unit 21 of the delayed light emission measuring device 1. It is preferable to use a mountain function as the function. A chevron-shaped function is a function that has a chevron shape, and the function value has a maximum value at a predetermined variable value, and is monotonously non-decreasing at a variable value below the predetermined variable value, greater than the predetermined variable value Is a monotonically non-increasing function.

で示されるローレンツ関数（コーシー分布）を用いることが考えられる。係数値ａ_ｉは時間方向への広がり、係数値ｂ_ｉは発光量が最大値を取る励起後時間ｔ、係数値ｃ_ｉは最大値の極大値に、それぞれ影響する。 As the plurality of functions, for example, a _i , b _i , c _i (i = 1, 2, 3) are coefficient values of the plurality of functions, t is a variable indicating an elapsed time (post-excitation time) from the reference time, Each time

It is conceivable to use the Lorentz function (Cauchy distribution) shown in FIG. The coefficient value a _i spreads in the time direction, the coefficient value b _i affects the post-excitation time t at which the light emission amount takes a maximum value, and the coefficient value c _i affects the maximum value of the maximum value.

となる。ここで、励起後時間ｔの基準時刻は、例えば、光源１２からの光の照射が終了した時刻から予め設定した所定の時間が経過した時刻とする。また、上記（２）の式において、右辺の第１項、第２項、第３項を、それぞれ時間軸上で最大値の現れる順番で、第１関数、第２関数、第３関数というように表す。すなわち、複数の関数の係数において、ｂ_１＜ｂ_２＜ｂ_３の関係が成立する。 In this case, the sum of multiple functions used for fitting is

It becomes. Here, the reference time of the post-excitation time t is, for example, the time at which a predetermined time has elapsed from the time when the light irradiation from the light source 12 is completed. In the expression (2), the first term, the second term, and the third term on the right side are referred to as the first function, the second function, and the third function in the order in which the maximum values appear on the time axis, respectively. Expressed in That is, the relationship of b ₁ <b ₂ <b ₃ is established among the coefficients of a plurality of functions.

Note that the function used in the fitting method is not limited to this, and other mountain functions such as a quadratic function and a normal distribution function may be used. Further, as long as it is a function that fits well, it does not have to be a mountain function. For example, a function represented by the following equation obtained by adding a coefficient d _i (i = 1, 2, 3) to the equation represented by (2) may be used.

  In order to perform the fitting method, an algorithm for performing the fitting is also stored in the recording unit 21 in advance. For example, a simplex method, a Gauss-Newton method, a Davidon Fletcher Powell method, a Brent method, a Monte Carlo method, and a simulated annealing method can be used as an algorithm for performing fitting.

  Then, an evaluation value is derived based on the coefficient value calculated by the fitting method. For example, the evaluation value is calculated based on an equation for calculating the evaluation value stored in the recording unit 21 in advance.

  By using such a fitting method, it is possible to derive an evaluation value objectively. In addition, since the time-dependent change in the light emission amount of delayed light emission is fitted to, for example, the equation shown in the above (2) or the equation shown in the above (3), the delayed light emission mechanism described with reference to FIG. Evaluation based on this becomes possible. Therefore, the oxidation-reduction state of the photosynthetic sample included in the evaluation sample can be appropriately evaluated.

  In the above embodiment, the standby time is adopted as the second irradiation condition that is changed when the measurement of the amount of delayed light emission is repeated a plurality of times, but the second irradiation condition to be changed is not limited to this. Since the redox state of the photosynthetic sample depends on the energy of the light applied to the photosynthetic sample and the efficiency of electron transfer of the photosynthetic sample, for example, at least of the light quantity, wavelength, pulse width, and irradiation time of the second excitation light One may be changed. These conditions regarding the second excitation light are closely related to the change in the redox state of the photosynthetic sample described with reference to FIG. Therefore, by changing at least one of these elements while repeating the first excitation light irradiation and the second excitation light irradiation a plurality of times, and deriving an evaluation value based on each measured light emission amount, The oxidation-reduction state of the photosynthetic sample contained in the evaluation sample can be appropriately evaluated.

  Further, the measurement may be repeated a plurality of times while changing environmental factors such as temperature and electric field that change the electron transfer efficiency of the photosynthetic sample. Furthermore, a plurality of oxidation-reduction states can be generated by using a combination of the standby condition and the above-described modification, or a combination of the above-described modifications. Therefore, the redox state of the photosynthetic sample included in the evaluation sample can be appropriately evaluated.

It is a schematic diagram which shows photosynthetic electron transfer. It is a figure which shows typically the delayed light emission measuring device which concerns on embodiment. FIG. 3 is a block diagram partially showing the delayed light emission measuring device shown in FIG. 2. It is a flowchart which shows the procedure of the evaluation method of a photosynthesis sample. It is a graph which shows the relationship between waiting time and the time-dependent change of delayed light emission. It is a graph which shows the relationship between the irradiation light quantity of excitation light, and the time-dependent change of delayed light emission. It is a graph which shows the relationship between the irradiation time of excitation light, and the time-dependent change of delayed light emission. It is a schematic diagram which shows the contrast of the oxidation reduction state in a state without saturation, and delayed light emission. It is a schematic diagram which shows contrast with a redox state and delayed light emission when the waiting time is not provided after 1st excitation light irradiation. It is a schematic diagram which shows contrast with a redox state and delayed light emission when standby time is provided for 2 second after 1st excitation light irradiation. 6 is a graph showing integrated values for the attenuation curve shown in FIG. 5, (a) is a graph for integrated value 1, (b) is for integrated value 2, and (c) is a graph for integrated value 3. FIG. It is a graph which shows the relationship between the waiting time in a standard sample, and the time-dependent change of the emitted light amount of delayed light emission. It is a graph which shows the relationship between the waiting time in an evaluation sample, and the time-dependent change of delayed light emission. 14 is a graph showing an integrated value based on the amount of delayed light emission shown in FIGS. 12 and 13, wherein (a) is an integrated value 1, (b) is an integrated value 2, and (c) an integrated value 3 is a graph. is there. It is a figure which shows the structure of the evaluation program which concerns on embodiment with a recording medium. FIG. 16 is a diagram showing a hardware configuration of a computer on which the evaluation program shown in FIG. It is the graph which showed the time-dependent change of the light emission amount of the delayed light emission about the standard sample in an Example. It is the graph which showed the time-dependent change of the light-emission quantity of the delayed light emission about the DCMU exposure sample in an Example. It is the graph which showed the time-dependent change of the light emission amount of the delayed light emission about the DBMIB exposure sample in an Example. It is the graph which showed the time-dependent change of the light emission amount of the delayed light emission about the Ant-A exposure sample in an Example. It is the graph which compared the integrated value of the standard sample in an Example, and the integrated value of a DCMU exposure sample, (a) is about integrated value 1, (b) is about integrated value 2, (c) is a graph about integrated value 3. It is. It is the graph which compared the integrated value of the standard sample in an Example, and the integrated value of a DBMIB exposure sample, (a) is about integrated value 1, (b) is about integrated value 2, (c) is a graph about integrated value 3. It is. It is the graph which compared the integrated value of the standard sample in an Example, and the integrated value of Ant-A exposure sample, (a) about integrated value 1, (b) about integrated value 2, (c) about integrated value 3 It is a graph of. It is a table | surface which shows value eII and vII in the standard sample in an Example and each exposure sample without saturation. It is a table | surface which shows the value vQ and% oQ of the standard sample in each Example, and each exposure sample. It is a table | surface which shows the value eI and cI in the standard sample in an Example and each exposure sample without saturation.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Delay light emission measuring device, 10 ... Measurement unit, 11 ... Sample installation part, 12 ... Light source, 13 ... Filter, 14 ... Condensing optical system, 15 ... Shutter, 16 ... Detection part, 16a ... Optical sensor, 16b ... Light quantity Calculation unit, 17 ... housing, 17a ... main body unit, 17b ... lid unit, 20 ... analysis unit, 21 ... recording unit, 22 ... calculation unit, 23 ... display unit, 24 ... input unit, 30 ... control unit, 31 ... Light source control unit, 32 ... detection control unit, 41 ... first cable, 42 ... second cable, 50 ... evaluation program, 51 ... light source control module, 52 ... detection control module, 53 ... calculation module, 60 ... recording medium, 70 …Computer.

Claims (9)

A method for evaluating a photosynthesis sample for evaluating an evaluation sample including a photosynthesis sample having a photosynthesis function,
A first excitation step of irradiating the evaluation sample with first excitation light under a predetermined first irradiation condition;
After the first excitation step, a second excitation step of irradiating the evaluation sample with second excitation light under a second irradiation condition having a small integrated light energy value as compared with the first irradiation condition;
After the second excitation step, a measurement step of measuring the amount of delayed luminescence generated from the evaluation sample;
An evaluation value is derived based on the light emission amount of delayed luminescence measured in the measurement step, and the evaluation step includes evaluating the evaluation sample based on the evaluation value and standard data.
In the measurement step, the second irradiation condition is changed to measure the light emission amount after the first excitation step and the second excitation step,
In the evaluation step, the evaluation sample is evaluated based on an evaluation value corresponding to the light emission amount measured in the measurement step and standard data corresponding to the evaluation value.   The standard data is a reference value, and in the evaluation step, the evaluation sample is evaluated based on an evaluation value corresponding to the light emission amount measured in the measurement step and the reference value corresponding to the evaluation value. The method for evaluating a photosynthetic sample according to claim 1.   3. The evaluation step according to claim 1, wherein the evaluation step derives an evaluation value based on an integrated value obtained by integrating the light emission amount of the delayed light emission measured in the measurement step every one or more measurement time zones. Evaluation method for photosynthetic samples.   The evaluation step derives coefficient values of the plurality of functions so that the temporal change in the light emission amount of delayed light emission measured in the measurement step is fitted as a sum of a plurality of preset functions, 3. The method for evaluating a photosynthetic sample according to claim 1, wherein an evaluation value is derived based on the evaluation value.   The second irradiation condition is a waiting time from the end of irradiation of the first excitation light to the start of irradiation of the second excitation light. The evaluation method of the photosynthetic sample as described in 2.   6. The photosynthesis according to claim 1, wherein the second irradiation condition is at least one of a light amount, a wavelength, a pulse width, and an irradiation time of the second excitation light. Sample evaluation method.   The said measurement step measures each said light-emission quantity after repeating the said 1st excitation step and the said 2nd excitation step in multiple times, changing the said 2nd irradiation conditions. The evaluation method of the photosynthetic sample as described in any one of these.   The method for evaluating a photosynthetic sample according to claim 1, wherein the evaluation step evaluates an environmental factor given to the evaluation sample. A photosynthesis sample evaluation program for evaluating an evaluation sample including a photosynthesis sample having a photosynthesis function,
Computer
First excitation means for irradiating the evaluation sample with first excitation light under a predetermined first irradiation condition;
After the first excitation means, second excitation means for irradiating the evaluation sample with second excitation light under a second irradiation condition having a small integrated light energy value as compared with the first irradiation condition;
Measuring means for measuring the amount of delayed luminescence generated from the evaluation sample after the second excitation means;
Deriving an evaluation value based on the amount of delayed luminescence measured by the measurement means, and functioning as an evaluation means for evaluating the evaluation sample based on the evaluation value and standard data,
The measuring means changes the second irradiation condition to measure the light emission amount after the first excitation means and the second excitation means,
The evaluation means evaluates the evaluation sample based on an evaluation value corresponding to the light emission amount measured by the measurement means and standard data corresponding to the evaluation value.

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