Protocol to study the photosynthetic response of maize to the CO₂-temperature coupling effect at ear stage using a specialized designed gradient

Summary

Global warming will change the photosynthesis and transpiration of plants greatly and ultimately affect water use efficiency (WUE). Here, we present a protocol to investigate the response of maize WUE to the coupling effect of CO₂ and temperature at ear stage using a specialized designed gradient. We describe steps for plant culture, parameter measurements, model fitting, and statistical analysis. This protocol holds potential for studying the response of WUE and CO₂ adaptation across various plant species.

For complete details on the use and execution of this protocol, please refer to Sun et al.¹

Graphical abstract

Highlights

• •Instructions for plant culture
• •Steps for designing specialized gradients to assess the effects of CO₂ and temperature
• •Procedures for parameter measurement, model fitting, and calculation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Global warming will change the photosynthesis and transpiration of plants greatly and ultimately affect water use efficiency (WUE). Here, we present a protocol to investigate the response of maize WUE to the coupling effect of CO₂ and temperature at ear stage using a specialized designed gradient. We describe steps for plant culture, parameter measurements, model fitting, and statistical analysis. This protocol holds potential for studying the response of WUE and CO₂ adaptation across various plant species.

Before you begin

Since 1750, due to the swift expansion of the global population and the widespread use of fossil fuels, there has been a rapid increase in the atmospheric concentration of carbon dioxide. Projections suggest that by the conclusion of the 21^st century,² this concentration will escalate from the current level of 280 μmol mol⁻¹ to 800 μmol mol⁻¹, intensifying the greenhouse effect. As per the IPCC AR6 report,³ the global average temperature is anticipated to climb by 1.5°C–2°C by the century's end compared to present levels. This temperature rise will directly impact physiological processes such as photosynthesis and transpiration in plants,⁴ thereby influencing Water Use Efficiency (WUE).

WUE, as defined by Gilbert et al.,⁵ represents the amount of CO₂ assimilation or dry matter production per unit mass of water consumed by plants. It serves as a crucial parameter for describing the water use characteristics of plant leaves and reflects their response to climate change. Given the real-world challenges of limited global water resources, population growth, and increasing food demand, enhancing WUE holds significant importance for agricultural production, ecological preservation, and development. However, current research on WUE often focuses on temperature or CO₂ as isolated factors.⁶ For instance, elevated CO₂ levels have been shown to enhance average WUE,^7,8 while high temperatures exert a linear negative impact on WUE.⁹ Yet, exploration of the coupled effects of these factors on WUE is limited.¹⁰

Here, we introduce a protocol aimed to investigate the response of maize WUE to the coupling influence of CO₂ and temperature during the ear stage, utilizing a specifically designed gradient. Detailed explanations of the gradient design, model fitting, and calculations are provided. This protocol offers a theoretical foundation for researching and implementing WUE enhancement strategies in maize amidst global warming concerns. Moreover, it holds promise for exploring WUE response and CO₂ adaptation across diverse plant species.

Plant culture

Timing: 10 weeks

Complete cultivation of plant experimental materials.

• 1.Plant 10 maize seeds in each open-air cultivation box filled with soil. Over a period of 10 weeks, administer 8 L of water to each box/seedling every 7 days, ceasing irrigation if rainfall exceeds 5 mm within the week.

Note: The experimental site was located in the Northwest A & F University (34°17′N, 108°4′E) in Yangling District, Xianyang City, Shaanxi Province, China, therefore, we selected Zea mays L. ‘Zhengdan 958’ as the experimental material, being the predominant maize variety in the Guanzhong area of Shaanxi Province. For the box and soil, the size of the boxes we used is 52 cm × 39 cm × 29.5 cm, and we filled them with local farmland topsoil. In case of weak seedling symptoms such as slow growth, thinness, deformity, paleness, yellowing, or premature leaf shedding, refer to problem 1.

Optional: The protocol below describes the specific steps for using ‘Zhengdan 958’. However, we have also used this protocol in ‘Jinkai 2’. Therefore, ‘Jinkai 2’ and other maize varieties can be used as alternative varieties.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
The seeds of “Zhengdan 958”	Yangling Junhui Seed Trading Company, China	N/A
Software and algorithms
SigmaPlot 14.0	Systat Software Inc., USA	https://grafiti.com/sigmaplot-detail/
Other
Open-air cultivation box (52 cm × 39 cm × 29.5 cm)	Mochuan flagship store, China	N/A
Local farmland topsoil	Shaanxi, Yangling, China	N/A
LI-6400	LI-COR Inc., USA	https://www.licor.com/env/support/LI-6400/manuals.html

Alternatives: The protocol use the LI-6400 (LI-COR Inc., USA) to measure the photosynthetic parameters, use the seeds of ‘Zhengdan 958’ to study and use Sigmaplot to create plots and analysis. Other portable photosynthetic apparatus, maize seeds and analytic plotting software could be used as replacements.

Step-by-step method details

Photosynthetic parameter measurement basis on CO₂ response curve

Timing: 6.5 h per day (from 8:30 to 11:30 and from 14:30 to 18:00) until the end of measurement stage, 3–7 days

Measure plant photosynthetic CO₂ response curves under five different temperature conditions and obtain the corresponding photosynthetic parameters for each condition.

• 1.
  Measure the photosynthetic-CO₂ response curve with the LI-6400 (LI-COR Inc., USA).

  • a.
    Before the measurement, equilibrate each plants for 10 min under the conditions of light intensity of 1200 μmol m⁻² s⁻¹, CO₂ concentration of 400 μmol mol⁻¹, temperature of (25 ± 0.2)°C and relative humidity of (71.2 ± 2.9)%.

    CRITICAL: Equilibrate the parameters under well-hydrated conditions as water deficiency can distort the vapor pressure deficit (VPD), which consequently impacts the stomatal conductance and transpiration (T_r). Ensuring equilibrium is reached is essential to prevent measurement instability.

  • b.
    Measure the photosynthetic-CO₂ response curve.

    • i.
      For the usage of LI-6400 (LI-COR Inc., USA), please follow https://licor.app.boxenterprise.net/s/s8zyqu2vwndny903qutg or https://www.licor.com/env/support/LI-6400/manuals.html.
    • ii.
      Use the LI-6400 temperature control system (LI-COR Inc., USA) to establish five temperature levels (20°C, 25°C, 30°C, 35°C, and 40°C) and set 11 CO₂ concentrations (400, 300, 200, 150, 100, 50, 400, 400, 600, 800, and 1000 μmol mol⁻¹, as shown in Figure 1). During measurements, maintain photosynthetically active radiation (PAR) in the leaf chamber at 1200 μmol m⁻² s⁻¹, while keeping other environmental factors unchanged in their natural state.

      Note: 20°C, 25°C, 30°C, 35°C and 40°C was set on the basis of the ambient temperature.

      The parameter measurement takes 120 s–200 s to reach steady state. The measurements for this study were conducted outdoors on sunny days, and the measurement environment may be influenced by the weather, potentially affecting the results.
    • iii.
      Use the LI-6400 to conduct the CO₂ response curve measurements on the widest part of the first fully expanded leaf of maize plants with similar growth status. Each leaf underwent two measurements, and the average value was taken as the result.

      Note: If there are any relevant problems with the use of LI-6400, please refer to problems 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11.

      CRITICAL: Schedule the photosynthetic-CO₂ response curve measurements between 8:30 and 18:00 to mitigate the influence of dim light on the experiment. Avoid measuring during noon due to the mid-day depression effect.

      Pause point: To ensure that the plant's growth status remains relatively unchanged, measurements should be conducted as compactly as possible. Subsequent model building and data analysis can be deferred.

Figure 1.

Schematic diagram of measurement sequence

Note: The bar represents the applied CO₂ concentration, and the arrowhead indicates the measuring direction. All authors permit the reuse of figures from the previous research article (Sun et al.¹).

Model fitting of the response of P_n and WUE to CO₂

Timing: Variable

Using the obtained photosynthetic parameter values, establish response models of P_n and WUE to CO₂, and acquire parameters that characterize CO₂ adaptation ability.

• 2.Use right-angled hyperbola modified model (Equation 1) to fit the CO₂ response curves of P_n at different temperatures.

$$P_n=a\frac{1-b{C}_a}{1+c{C}_a}{C}_a-d$$ (Equation 1)

Use Equations 2, 3, and 4 to estimate the CSP, A_max and CCP respectively.

$$\text{CSP}=\frac{\sqrt{\left(b+c\right)/b}-1}{c}$$ (Equation 2)

$$A_{\mathit{max}}=a{\left(\frac{\sqrt{b+c}-\sqrt{b}}{c}\right)}^2-d$$ (Equation 3)

$$\text{CCP}=\frac{a-cd-\sqrt{a^2-2ad\left(2b+c\right)+c^2{d}^2}}{2ab}$$ (Equation 4)

Note: Due to its superior fitting capability for plant response curves during activation, inhibition, and adaptation stages, the modified right-angled hyperbola model enables direct calculation of CSP, CCP, and A_max. Therefore, this protocol uses the model to fit the CO₂ response curves of P_n at various temperatures. Where P_n is the net photosynthetic rate, a is the initial carboxylation efficiency of CO₂ response curve, C_a is the atmospheric CO₂ concentration, and b, c and d are the model evaluation coefficients (the units of b and c are mol μmol⁻¹, and the unit of d is μmol m⁻² s⁻¹). In Equation 2, obtain CSP by solving the first derivative of Equation 1 and calculating the solution when its derivative value is 0; In Equation 3, Obtain A_max by substituting the Equation 2 into the Equation 1; In Equation 4, Obtain CCP by substituting P_n = 0 into Equation 1.

Optional: The protocol below describes the specific steps for calculating CSP, A_max and CCP by Equations 2, 3, and 4. However, we have also used another protocol to estimate these parameters. Use Linear fitting (Equation 5) to fit with P_n in the range of 600, 800 and 1000 μmol mol⁻¹, and P_n in the range of 400 μmol mol⁻¹ ∼ 400 μmol mol⁻¹ (1^st ∼ 6^th and 8^th CO₂ concentration) respectively (Figure 2):

$$P_n=k{C}_a-d$$ (Equation 5)

Figure 2.

Schematic diagram of linear fitting method

Note: Points are the average measurements of the photosynthetic CO₂ response at any set of test temperatures, and two straight lines are fitted by data before 400 μmol mol^-1 and data after 400 μmol mol^-1 respectively. The intersection of the straight line fitted by data before 400 μmol mol^-1 and the X axis is CCP; the abscissa of the intersection of two straight lines is the estimated value of CSP, and the ordinate is the estimated value of A_max. All authors permit the reuse of figures from the previous research article (Sun et al.¹).

In Equation 5, k is the parameter to be estimated, and other parameters use the same definition as Equation 1. The abscissa of the intersection of two straight lines is the estimated value of CSP, and the ordinate is the estimated value of A_max. The intersection of the fitting curve of 400 μmol mol⁻¹–400 μmol mol⁻¹ and the positive semi-axis of X axis is the estimated value of CCP.

• 3.Use piecewise fitting quadratic function to get the response curve of WUE (Equation 6):

$$\text{WUE}=a{C_a}^2+b{C}_a+c$$ (Equation 6)

Note: To assess plant adaptability to elevated CO₂, the CO₂ concentration setup in this study differs from the normal CO₂ response curves. This variation in the CO₂ concentration gradient we specially designed resulted in discontinuous WUE responses to CO₂, significant disparities in numerical values at breakpoints, and a coefficient of determination (R²) approaching zero when fitting the right-angled hyperbolic correction model. Therefore, the quadratic function piecewise fitting is carried out for WUE, the first stage is 0–400 μmol mol⁻¹, and the second stage is 400–1000 μmol mol⁻¹, as T_r is also different before and after the segmentation, so this protocol only calculate the slope of T_r but not fit the model. In Equation 6, C_a is the atmospheric CO₂ concentration, and a, b and c are the model evaluation coefficients (a and b have the unit of mol μmol⁻¹, and the unit of c is (μmol CO₂ mmol H₂O⁻¹)).

• 4.
  Use the ratio between the difference of physiological parameters to describe the ability of CO₂ adaptation.

  • a.
    Use the ratio between the difference of physiological parameters between 7^th and 6^th CO₂ concentration and the maximum physiological parameters in the fitting curve as the index of CO₂ adaptation (I_ac, Equation 7).

    $$I_{ac}=\frac{{\Delta P_n}_{\left(7-6\right)}}{A_{\mathit{max}}}$$ (Equation 7)
  • b.
    Use the ratio between the difference of physiological parameters between 8^th and 7^th CO₂ concentration and the maximum physiological parameters in the fitting curve to take as the index of CO₂ secondary adaptation (I_acII, Equation 8).

    $$I_{ac\text{II}}=\frac{{\Delta P_n}_{\left(8-7\right)}}{A_{\mathit{max}}}$$ (Equation 8)

    Note: The I_ac assesses plant adaptability to elevated CO₂, with the protocol subtracting photosynthetic parameter values of the 6^th (low, 50 μmol mol⁻¹) from the 7^th (environmental, 400 μmol mol⁻¹) CO₂ concentrations. Meanwhile, the I_acII evaluates continuous adaptability with CO₂ maintained at a certain level, subtracting values of the 7^th CO₂ concentration (400 μmol mol⁻¹) from the 8^th (400 μmol mol⁻¹). Equation 7 defines I_ac as the CO₂ adaptation index, representing the difference in P_n between the 7^th and 6^th CO₂ concentrations; Equation 8 defines I_acII as the CO₂ secondary adaptation index, representing the difference in P_n between the 8^th and 7^th CO₂ concentrations. A_max is the maximum P_n value, and T_r and WUE follow the same calculation method as in Equations 7 and 8.

Expected outcomes

The protocol is expected to produce the CO₂ response curve of P_n, T_r and WUE, where the WUE will be shown as a piecewise function (Figures 3, 4 and 5). This protocol is also expected to produce graphs that could be used to evaluate the adaptability of plants to elevated CO₂ and to evaluate the continuous adaptability of plants to CO₂ (Figure 6).

Figure 3.

Response of P_n to CO₂ and temperature in ‘Zhengdan 958’

Response of P_n to CO₂ (A) and A_max to temperature (B) in ‘Zhengdan 958’.

Note: All authors permit the reuse of figures from the previous research article (Sun et al.¹).

Figure 4.

Response of T_r to CO₂ and temperature in ‘Zhengdan 958’

Response of T_r to CO₂ (A, B) and T_rmax to temperature (C) in ‘Zhengdan 958’.

Note: The bar represents the applied CO₂ concentration, and the symbols of open and closed circles, open and closed squares, and open triangles represent 20°C, 25°C, 30°C, 35°C and 40°C in order. All authors permit the reuse of figures from the previous research article (Sun et al.¹).

Figure 5.

Response of WUE to CO₂ and temperature in ‘Zhengdan 958’

Response of WUE to CO₂ (A: 0–400 μmol mol⁻¹; B: 400–1000 μmol mol⁻¹) and WUE_max to temperature (C) in‘Zhengdan 958’.

Note: All authors permit the reuse of figures from the previous research article (Sun et al.¹).

Figure 6.

Adaptive Response of P_n, T_r and WUE of ‘Zhengdan 958’ to CO₂

Adaptive Response of P_n (A), T_r (B) and WUE (C) of ‘Zhengdan 958’ to CO₂.

Note: The bar represents the applied CO₂ concentration, and the symbols of open and closed circles, open and closed squares, and open triangles represent 20°C, 25°C, 30°C, 35°C and 40°C in order. All authors permit the reuse of figures from the previous research article (Sun et al.¹).

Quantification and statistical analysis

• 1.
  Use Sigmaplot 14.0 to create plots and analyze, and test the normality of data sets via Shapiro-Wilk test.

  • a.
    Open the Sigmaplot 14.0, click ‘Create new blank notebook’.
  • b.
    Input the data in the blank, and create the graph.

    • i.
      Select all data, and click ‘Create Graph’.
    • ii.
      Click ‘Scatter’ - ‘Simple Scatter’.
    • iii.
      Choose ‘XY pair’, then choose ‘Complete’.
  • c.
    Right click on any point in the plot, and choose ‘Dynamic curve fit’.
  • d.
    Create and use the right-angled hyperbola modified model.

    • i.
      Set the equation category as ‘User-Defined’, choose ‘new’.
    • ii.
      Set the equation as ‘f = a∗(1−b∗x)∗x/(1 + c∗x)−d fit f to M’, and hit ‘Enter’ between ‘f = a∗(1−b∗x)∗x/(1 + c∗x)−d’ and ‘fit f to M’.
    • iii.
      Set the variables as ‘x = col(1) M = col(2)’, and hit ‘Enter’ between ‘x = col(1)’ and ‘M = col(2)’.
    • iv.
      Set the initial parameters as ‘a = 1 b = 1 c = 1 day = 1’, and hit ‘Enter’ between ‘a = 1’, ‘b = 1’, ‘c = 1’ and ‘d = 1’ respectively.
    • v.
      Set the constraints as ‘a>0 b > 0 c > 0 d > 0’, and hit ‘Enter’ between ‘a>0’, ‘b > 0’, ‘c > 0’ and ‘d > 0’ respectively, then choose ‘OK’ and ‘Finish’.
    • vi.
      Get the report, and analyze the data.

Note: After creating the model, just choose the user-defined model and choose ‘Finish’ instead of ‘new’. When analysing the data, use the a, b, c, d and p [Normality Test (Shapiro-Wilk)] in the report with Equations 2, 3, and 4, and the other models in this protocol could be created in the same way. If the p value couldn’t pass the Shapiro-Wilk test or R² is lower than 0.85, refer to problem 12.

CRITICAL: The constraints must be set as ‘a>0 b > 0 c > 0 d > 0’ to ensure that the equation adheres to physiological significance.

Optional: After setting appropriate constraints, the initial values for fitting had minimal impact on model establishment. Therefore, we simply set all initial fitting values to "1". You can substitute "1" with any number within the range allowed by the constraints that you believe may be close to the final result.

Sample data set:

CO2 (μmol mol−1)	Pn (μmol m−2 s−1)
400	11.33
300	9.48
200	7.18
150	5.88
100	4.27
50	3.37
400	11.16
600	12.25
800	12.75
1000	13.23

Limitations

Insufficient water supply during the plant culture stage, insufficient balance before measuring, and measuring the photosynthetic parameters during the time except for 8: 30 to 11: 30 and 14: 30 to 18: 00 may produce dissimilar results.

Troubleshooting

Problem 1

Weak seedling phenomenon occurs (Related to Plant culture 1).

Potential solution

Provide plants with the light intensity no less than 1200 μmol m^-2 s^-1. We did not apply fertilizer during the experiment, but it can add as appropriate if required.

Problem 2

Temperature anomaly response (Related to Step 1).

Potential solution

If T_block, T_air, and T_leaf are negative, please check the hardware connections. If the issue is not resolved, it may be necessary to send the device back for repairs.

If only the T_leaf temperature is negative, please replace the leaf temperature thermocouple.

If there is zero-point drift in the leaf temperature thermocouple, and the temperature difference between T_block and T_leaf is not within 0.1°C, perform temperature calibration using a screwdriver on the potential adjuster at the bottom of the analyzer.

Problem 3

Abnormal response of net photosynthetic rate (Photo) value (Related to Step 1).

Potential solution

The abnormal response of the Photo value can be categorized into two types: Photo value instability and Photo value anomaly.

Photo value instability may be caused by the instability of CO₂R or CO₂S. Check if the soda lime tube is in a complete Scrub state, ensure the soda lime is effective, and inspect if the leaf chamber is fully sealed.

In the case of Photo value anomaly, verify if CO₂ concentration is normal, the light source is functioning correctly, the air path is not blocked, and the leaf area input is accurate. When the Photo value doesn’t change with CO₂ concentration, indicating that the photosynthetic response of the leaves is not transmitted to the sample chamber, assess the leaf chamber fan’s operation by listening for changes in sound when toggling it; if there is no change, it indicates fan malfunction and requires replacement.

Problem 4

Transpiration rate (T_r) is a negative value (Related to Step 1).

Potential solution

Check if the leaf area input is correct, and verify that H₂OS is less than H₂OR. If H₂OS is less than H₂OR, tighten the desiccant tube in the Scrub direction and the soda lime tube in the Bypass direction. Close the empty leaf chamber and wait for approximately 5 min. Examine rows a for H₂OR and H₂OS, which should be within ±0.5 μmol mol⁻¹; if they are not within this range and show a decreasing trend, it is advisable to wait for an additional 10 min. If the values still exceed the specified range, consider restoring to factory settings using the following steps:

On the main screen, press F3 (Calib Menu), choose View Settings, and press enter;

Select View History, press enter, press the Home key to choose the "at factory" in the first row;

Press F3 (Set to), F5 (OK); then press the up arrow, select View Current, press enter;

Press F3 (Save), press Y, press enter, and finally, press F5 (Done).

Problem 5

Stomatal conductance (Cond) value is abnormal (Related to Step 1).

Potential solution

If the leaf’s stomatal conductance (Cond) value is not within the range of 0–1, first check if the transpiration rate is a negative value (Refer to the potential solution of problem 4). If the transpiration rate is normal, inspect the leaf temperature thermocouple for proper functioning (Refer to the potential solution of problem 2).

Problem 6

The intercellular CO₂ concentration (C_i) value is abnormal (Related to Step 1).

Potential solution

An excessively low or negative intercellular CO₂ concentration (C_i) value is often caused by stomatal conductance. First, check if the stomatal conductance (Cond) value is normal. If the stomatal conductance value is normal, it might be caused by an excessively high net photosynthetic rate, and it is recommended to recalibrate the IRGAs.

Problem 7

IRGAs Not Ready (Related to Step 1).

Potential solution

Check if the IRGAs have been powered on for a sufficient warm-up time (about 10 min).

Ensure proper cable connections: align the red mark on the circular plug with the one on the analyzer, push it in horizontally until fully inserted, and listen for a locking sound.

Examine the first two parameters (CRagc and CSagc) in the L row: if CRagc exceeds 1500, the reference chamber optics may be dirty; If CSagc exceeds 1500, the sample chamber optics may be dirty (follow the cleaning procedure at https://licor.app.boxenterprise.net/s/s8zyqu2vwndny903qutg or https://www.licor.com/env/support/LI-6400/manuals.html).

If the issue persists after addressing these factors, return the equipment for repair.

Problem 8

High humidity alarm (Related to Step 1).

Potential solution

When the instrument’s humidity reaches 95%, it triggers a high humidity alarm. In such cases, you can rotate the adjustment knob of the desiccant tube towards the Scrub direction, increase the flow rate moderately, or raise the temperature of the T_block in the cooler.

If the instrument issues a high humidity alarm at a relatively low relative humidity of 60–70%, it indicates potential issues such as cable disconnection between the console and the analyzer, aging of the leaf chamber mixing fan, etc. In such situations, it is advisable to return the instrument for repairs.

Problem 9

Abnormal operation of the light source (Related to Step 1).

Potential solution

Setting light intensity, but the light source is not on: Check if the power supply cable for the red-blue light source’s black plug is properly connected.

Setting light intensity, the light source is on, but PAR_in is 0, and the main interface shows no light source signal: Please check if the plug of the built-in light quantum sensor is properly connected.

Problem 10

Airway blockage (Related to Step 1).

Potential solution

In the measurement menu, press 2, F2, set the Target flow rate to 1000. Check if the flow rate (b row) can reach 650 or above (this value is a reference at standard atmospheric pressure; it may decrease at higher altitudes). If not, it indicates an airway blockage. Here’s the troubleshooting:

Detach the soda lime tube/desiccant tube separately and check if the flow rate is normal. If normal, it indicates a blockage in the corresponding tube.

If the flow rate in Bypass mode is abnormal, blow into the air inlet with an ear syringe to check for blockage. If the air inlet is normal, it suggests a blockage in the upper plastic tubing, requiring a replacement.

If the flow rate in Bypass mode is normal but Scrub mode is abnormal, replace the filter.

If both tubes are normal, inspect the filters and air inlet path inside the console.

Problem 11

Chemical reagent failure (Related to Step 1).

Potential solution

Desiccant: Fresh and effective desiccant is blue, and it turns pink when absorbing moisture. Replace the desiccant in the desiccant tube when 2/3 of it has turned pink. During replacement, ensure the threads are clean and dry to prevent gas leaks.

Soda Lime: Rotate the soda lime tube to the complete Scrub. After observing a decrease in CO₂R to the minimum, blow air into the inlet. If the CO₂R reading fluctuates by more than 2 μmol mol⁻¹, it indicates soda lime failure, requiring replacement.

Problem 12

The p value couldn’t pass the Shapiro-Wilk test or R2 is lower than 0.85 (related to Step 5).

Potential solution

Try to measure the CO₂ response curve of another plants in the same treatment group, if not work, try to balance for longer time before measuring or check if there is some problems in the plant culture stage.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yongsheng Wei (wysh70@nwafu.edu.cn).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Sicong Sun (ss_717907@nwafu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate any code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.