Photosynthetic probe fluorescence measurement technology MPF--a new method for accurately obtaining Fm

Accurate measurement of the maximum fluorescence yield under light Fm 'is the focus and difficulty of measuring the fluorescence parameters of plant leaves. Inaccurate measurements Fm 'will result in an error range calculation associated fluorescence parameters, such as non-photochemical quenching NPQ mesophyll conductance g _m and the like.

Many studies have shown that Fm' will increase with increasing "saturation flash" intensity. PSII has a fast turnover capability, and the traditional "saturation flash" technology cannot completely reduce the receptor side of the PSII reaction center, so it is easy to cause an underestimation of the maximum fluorescence yield Fm' under light. The solution is to first measure the apparent ^A Fm ' under a number of different "saturation flash" gradients, and then establish the inverse of the "saturation flash" intensity and its linear equation relationship, and calculate the corresponding when the "saturated flash" tends to infinity. Fluorescence yield Fm' (Reference: Earl, H. And Ennahli, S. Estimating photosynthetic electron transport via chlorophyll fluorometry without Photosystem II light saturation. Photosynthesis Research, 2004, 82: 177~186. ). This method is very time consuming, thus limiting its application in actual measurements.

Moreover, it is necessary to assume that the process rate constants (such as kF, kD, etc.) remain unchanged and do not trigger side reactions when using traditional "saturation flash" techniques, and many studies have shown that the above assumptions are not true under high-intensity "saturation flash". .

In response to these shortcomings of the traditional "saturation flash", the technical team of LI-COR of the United States launched a new technology Multiphase Flash ^TM in 2009. For details, please see:

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Multiphase Flash ^TM flash multiphase technology (~ 1s) quickly measured during a brief flash plurality of flash intensity and fluorescence yield corresponding thereto ^A Fm ', by a linear fit accurately calculate ^E Fm'. Experimental results show:

(1) Under different "saturated flash" intensities, the difference in ^E Fm' estimated by the Multiphase Flash ^TM method is not significant and always higher than that measured by conventional methods. The ΦPSII calculated by ^E Fm' can be 15-30% higher than the traditional "saturation flash" technology measurement result;

(2) A linear equation is established for the electron transfer rate J calculated by ^E Fm′ and the net photosynthetic rate A _G , and the slope is 4.7±0.2, that is, about 4.7 electrons are fixed by one CO ₂ , which is consistent with the theoretical value. The LI-COR expert group published an article in Plant Cell & Environment in 2013: " Loriaux SD , Avenson TJ, Welles JM, etc. Closing in on maximum yield of chlorophyll fluorescence using a single multiphase flash of sub-saturating intensity. Plant, Cell & Environment, 2013, 36(10) : 1755 ~1770. ".

And for the fact that the traditional "saturation flash" technology measurement can not avoid triggering side reactions, Multiphase Flash ^TM technology can calculate ^E Fm ' under low intensity "saturated flash", as shown in the figure below, the light intensity is 4500 to 9000 μmolm ^-2 s ^-1 . The ^E Fm' obtained by the Multiphase Flash ^TM multiphase flash technology remains essentially unchanged; while the ^A Fm' obtained using the traditional "saturated flash" technique continues during the process of "saturated light intensity" from 4500 to 9000 μmol m ^-2 s ^-1 Increase, not really "saturated". Visible, Multiphase Flash ^TM multiphase technology can flash in a low intensity "saturated Flash" lower, as 4500μmolm ^-2 s ^-1, a more realistic estimate ^E Fm ', which reduces the maximum degree of saturation because too high intensity flash results The occurrence of side reactions is therefore very suitable for fluorescence measurements of plants that may cause light damage.

The difference between the traditional " saturation flash " and Multiphase Flash ^TM multiphase flash technology is shown in the figure below:

The traditional "saturation flash", also known as the rectangular flash (RF) measurement method: hit a saturated flash (Q) for about 400 to 1200 milliseconds, during which the fluorescence value is continuously recorded, and the highest fluorescence value is selected as ^A Fm'.

Flash technology multiphase Multiphase Flash ^TM three phases: (1) a saturated hit flash (Q), a duration of about 300 ms for reduction QA-PQ base; (2) a constant speed of about 300 milliseconds Q value decreases; ( 3) Return to the initial high Q value, hold for about 300 milliseconds, check for non-photochemical quenching (QN) caused by flash

According to the Fm' value obtained in the second stage and the 1E4/Q regression analysis to estimate the maximum fluorescence value when the intensity is infinite.

Multiphase Flash ^TM multi-phase flash technology is superior to other traditional evidence "saturation flash":

Use multiphase Multiphase Flash ^TM Flash technology, i.e., curve fitting method of obtaining the intercept (intercept method) estimated maximum, the real ^E Fm ', the ^E Fm' electron transfer rate was calculated to J and the total CO ₂ assimilation rate The efficiency relationship between the two is true, meeting the theoretical requirements, and using the traditional saturated flash single pulse method to obtain the apparent maximum fluorescence yield ^A Fm ', the electron transfer rate and total calculated from ^A Fm ' The relationship between the CO ₂ assimilation rates is deviated from the theory, significantly less than 4, as shown in the following figure, the left picture is described in the article published by Loriaux SD on Plant Cell & Environment in 2013; the right picture is Earl H. in 2004. The experimental conclusions published in Photosynthesisi Research.

Quantitative relationship between electron transfer rate J and total assimilation rate of CO ₂

(Theoretical maximum efficiency: 4 electrons fixed 1 CO ₂ )

Using Flash technology multiphase Multiphase Flash ^TM obtained ^E Fm 'calculated mesophyll conductance g _m over conventional "flash saturated" obtained by the method ^A Fm' calculated conductance g _m mesophyll more realistic and reasonable, below: