Leaf Photosynthesis MultispeQ Chloroplast movement JV1.0


Summary

Measures many photosynthesis-related parameters in <15 seconds, including:

  • Chlorophll Fluorescence: Phi2, PhiNPQ, PhiNO, NPQt, qL, LEF
  • Relative Chlorophyll: SPAD
  • Proton Motive Force: ECSt, vH+, gH+
  • Absorbance at: 450, 535, 605, 650, 730, 850, 880, and 940nm.
  • Leaf Thickness (in mm), angle, and cardinal direction
  • Leaf Temperature and differential from ambient temperature
  • Environmental conditions: PAR and ambient temperature/pressure/humidity

Description

It's not practical to fully describe what all of these measurements mean here. If you want to learn more, a good place to start is this video: https://www.youtube.com/watch?v=pU5vOtE1wE8 . Check the PhotosynQ youtube channel for other videos explaining photosynthesis and related parameters.

This article is also helpful http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3040.2007.01680.x/abstract. It's paywalled, but email admin@photosynq.org to request a copy.

If you want to see the exact calculations for each parameter, please see the associated macro called "Leaf Photosynthesis MultispeQ V1.0".

Finally, there are two publications about the MultispeQ Beta device covering many of the below parameters and comparisons to commercial instruments. While that is technically an older model device, the calculations and comparisons are still true for this protocol. You can find them at the Royal Society for Open Science, and .

Works with the MultispeQ V1.0 device only

The Leaf Photosynthesis protocol collects many different measurements of a leaf as quickly as possible. It attempts to be both accurate and fast, so that large amounts of data can be collected quickly. There are 3 main components of the protocol which we will describe in detail.

Click on this link to see an example measurement - https://photosynq.org/projects/disease-damage-and-drought-impacts-on-photosynthesis/explore/363687

1) Proton Motive Force

Proton Motive Force is the chemical and electrical gradient of protons (H+) across the thylakoid. Protons can move through the ATP synthase when passing from inside to outside the thylakoid membrane, providing energy to convert ADP to ATP as they pass. The capacity and flow rate of protons can be regulated by the plant in response to environmental conditions.

Proton Motive Force is measured in the MultispeQ by measuring the electrochromic shift of carotenoids in the light harvesting complex. This is a really interesting effect I strongly suggest you look up. The short description is when carotenoids (or any polar molecule) are stuck (not floating around in water or something), their absorbance shifts slightly if they are placed under an electric field. In our case, there are carotenoids sitting on the thylakoid membrane, and as positively charged protons build up in the thylakoid those carotenoids experience a larger and larger electric field, causing a shift in absorbance. As the positively charged protons move out of the ATP synthase, the electric field is reduced and the absorbance of the carotenoids shifts back to where it was.

This protocol measures that shift in absorbance of carotenoids using a green (545nm) LED from the plants normal state (ambient conditions) to darkness back to ambient. This short dark period causes all of the protons to empty out of the thylakoid which we can see as a shift in absorbance.

The maximum rate of protons exiting the ATP synthase (called rate of flux, or vH+), the total quantity of protons which exited (called ECSt) and the time until half of those protons exited (called gH+) are measured. Of all those parameters, gH+ is the most comparable across plants, as the other two are affected by leaf thickness, chlorophyll density, and other factors.

You can see the effect by zooming in on the first part of the trace. The ECS effect is most visible in high light conditions (because the thylakoid is going from a high density of protons to darkness, causing a large shift in the absorbance) and looks something like an extended shark's fin. Remember that this trace is showing transmission, not absorbance, though it could be transformed to show absorbance if you want to.

2) Chlorophyll Fluorescence

The second part of the trace uses chlorophyll fluorescence to understand Photosytem II. Chlorophyll fluorescence occurs when visible light absorbed by the plant is re-radiated in the near infra-red range (about 680 - 700nm), and is an important mechanism for plants to dissipate unwanted energy. By measuring chlorophyll fluorescence under ambient conditions (Fs) and high light (Fm'), and estimating fluorescence under dark conditions (Fo') by applying far red light to run photosystem I and fully reducing photosystem II, we can then calculate the amount of energy from photosystem II which goes to photosynthesis (Phi2), dissipated as heat via non-photochemical quenching (PhiNPQ), and all other places (PhiNO).

Wikipedia has good entries describing existing instruments and outputs which we won't repeat here:

https://en.wikipedia.org/wiki/Chlorophyll_fluorescence#Chlorophyll_fluorometers

3) Absorbance + Relative Chlorophyll

Absorbance can identify compounds which have a specific color. Chlorophyll is the most obvious example, but anthocyanins (purple/blue), carotenoids (orange), and flavenoids (pinkish, like those found in tomato skins, for example) can also be measured using absorbance.

It's important to remember that the visible color of a compound is what is most reflected (lowest absorbance), but to measure absorbance we need the color which is most absorbed. For example, chlorophyll is measured using absorbance in the red region (where it most absorbs), even though it's visibly green (where it most reflects). This can be counterintuitive but makes sense when you walk through the logic.

Also, to measure absorbance we need a blank. A blank measures how much light passes through no sample (ambient air).

Let's walk through the calculations relating to absorbance.

Transmittance (Ts) is the proportion of light which passes through the sample (0% - 100%) relative to a blank (no sample).

Ts = sample / blank

Ts            transmittance of sample
sample        raw detector value from sample
blank         raw detector value from blank

Absorbance is the opposite of transmission, so we need to do a bit more math.

_abs = -1*log(Ts)

abs         absorbance of sample
Ts          transmittance of sample

In order to convert a raw absorbance value to a relative concentration, the thickness of the sample must be estimated. Otherwise, a low concentration but very thick sample and a high concentration very thin sample would produce similar absorbance readings. We can estimate thickness by using near infra-red (940nm) which is not absorbed by the sample (we assume, that's a simplifying and imperfect assumption), but is reflected and refracted based on the sample thickness. So lower near infra-red tranmission, thicker sample.

I'm going to call this thickness-adjusted absorbance value SPAD because that's what it's called historically when measuring chlorophyll (see Minolta SPAD meter for example). So the final formula to account for this thickness using near infra-red is:

SPAD = 100*log(Ts / Tir)

SPAD           thickness adjusted absorbance
Ts             sample transmittance
Tir            near infra-red (940nm) transmittance

In the MultispeQ, this usually outputs a value between 0 - 125 for any given wavelength. For relative chlorophyll content, we then normalize our values to a Minolta SPAD 502+ to make it more easily comparable and compatible to users. This range is 0 - 75.

Finally, we have to take one additional step due to limitations of the hardware. The detector (pin photodiode) which is used to measure the light for any given absorbance measurement has a limited range - too much light can max out the signal. We can adjust the light intensity, pulse length, and pulse size in order to adjust that range to avoid maxing out the detector. Let's call this process adjusting the gain. However, for any given leaf, we can't know ahead of time how much to adjust the gain - thicker leaves will require high intensities and longer pulses, while thinner leaves require lower intensities and shorter pulses.

In order to address this problem, we take 3 absorbance measurements at 3 gain settings for each sample - a low, medium, and high gain for thin, medium, and thick leaves. Then, after the measurement, the macro automatically chooses the most appropriate gain setting based on the response (so skip the setting where the signal is maxed out or too low).

To sum up We measure absorbance on 8 lights (450, 530, 605, 650, 730, 850, 880, 940) at 3 gain settings (24 total). For the visible lights (450, 530, 605, 650, and 730) we apply the SPAD calculation to get a thickness-normalized value. We apply an additional normalization for the SPAD for red to make the range similar to that of a Minolta SPAD 502+. We then choose the values which are within an acceptable range (not too high, not too low) from the 3 intensities we measured.

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      [
        1
      ],
      [
        1
      ],
      [
        1
      ],
      [
        0
      ],
      [
        3
      ],
      [
        3
      ],
      [
        3
      ],
      [
        3
      ],
      [
        3
      ],
      [
        3
      ],
      [
        3
      ],
      [
        0
      ],
      [
        3
      ],
      [
        1
      ],
      [
        2
      ],
      [
        3
      ],
      [
        4
      ],
      [
        6
      ],
      [
        8
      ],
      [
        9
      ],
      [
        10
      ],
      [
        1
      ],
      [
        2
      ],
      [
        3
      ],
      [
        4
      ],
      [
        6
      ],
      [
        8
      ],
      [
        9
      ],
      [
        10
      ],
      [
        1
      ],
      [
        2
      ],
      [
        3
      ],
      [
        4
      ],
      [
        6
      ],
      [
        8
      ],
      [
        9
      ],
      [
        10
      ],
      [
        4
      ],
      [
        0
      ],
      [
        4
      ],
      [
        0
      ],
      [
        4
      ],
      [
        0
      ],
      [
        1
      ],
      [
        1
      ],
      [
        1
      ],
      [
        0
      ],
      [
        3
      ],
      [
        3
      ],
      [
        3
      ],
      [
        3
      ],
      [
        3
      ],
      [
        3
      ],
      [
        3
      ],
      [
        0
      ],
      [
        3
      ],
      [
        1
      ],
      [
        2
      ],
      [
        3
      ],
      [
        4
      ],
      [
        6
      ],
      [
        8
      ],
      [
        9
      ],
      [
        10
      ],
      [
        1
      ],
      [
        2
      ],
      [
        3
      ],
      [
        4
      ],
      [
        6
      ],
      [
        8
      ],
      [
        9
      ],
      [
        10
      ],
      [
        1
      ],
      [
        2
      ],
      [
        3
      ],
      [
        4
      ],
      [
        6
      ],
      [
        8
      ],
      [
        9
      ],
      [
        10
      ]
    ],
    "open_close_start": 1,
    "recall": [
      "colorcal_blank1[1]",
      "colorcal_blank1[2]",
      "colorcal_blank1[3]",
      "colorcal_blank1[4]",
      "colorcal_blank1[6]",
      "colorcal_blank1[8]",
      "colorcal_blank1[9]",
      "colorcal_blank1[10]",
      "colorcal_blank2[1]",
      "colorcal_blank2[2]",
      "colorcal_blank2[3]",
      "colorcal_blank2[4]",
      "colorcal_blank2[6]",
      "colorcal_blank2[8]",
      "colorcal_blank2[9]",
      "colorcal_blank2[10]",
      "colorcal_blank3[1]",
      "colorcal_blank3[2]",
      "colorcal_blank3[3]",
      "colorcal_blank3[4]",
      "colorcal_blank3[6]",
      "colorcal_blank3[8]",
      "colorcal_blank3[9]",
      "colorcal_blank3[10]",
      "colorcal_intensity1_slope[2]",
      "colorcal_intensity1_yint[2]",
      "colorcal_intensity2_slope[2]",
      "colorcal_intensity2_yint[2]",
      "colorcal_intensity3_slope[2]",
      "colorcal_intensity3_yint[2]",
      "ir_baseline_slope[5]",
      "ir_baseline_yint[5]",
      "ir_baseline_slope[3]",
      "ir_baseline_yint[3]"
    ],
    "environmental": [
      [
        "detector_read1",
        5,
        700,
        1,
        30
      ],
      [
        "detector_read2",
        1,
        150,
        3,
        15
      ],
      [
        "light_intensity"
      ],
      [
        "temperature_humidity_pressure"
      ],
      [
        "temperature_humidity_pressure2"
      ],
      [
        "contactless_temp"
      ],
      [
        "thickness"
      ],
      [
        "compass_and_angle"
      ]
    ],
    "averages": 1
  }
]
Default avatar
Created by

Judy Malas


Category

Plants

Compatible Instruments

MultispeQ v1.0 MultispeQ v2.0


Latest Update

Jan 2018