Getting Started With PhotoGEA

Overview

PhotoGEA (short for photosynthetic gas exchange analysis) is an R package that provides a suite of tools for loading, processing, and analyzing photosynthetic gas exchange data.

Installing PhotoGEA

The easiest way to install PhotoGEA is to type the following from within the R terminal:

remotes::install_github('eloch216/PhotoGEA')

Note that this method requires the remotes package, which can be installed from within R by typing install.packages('remotes').

An Example: C₃ CO₂ Response Curves

As an example, we will read data from two Licor Li-6800 log files that contain several A-Ci curves measured from tobacco and soybean plants, fit a model to each response curve, and then plot some of the results. This is a basic example that just scratches the surface of what is possible with PhotoGEA.

(Note: When loading your own files for analysis, it is not advisable to use PhotoGEA_example_file_path as we have done in the code below. Instead, file paths can be directly written, or files can be chosen using an interactive window. See the Analyzing C3 A-Ci Curves vignette for more information.)

Fitting the Curves

The following code can be used to read the data and fit each curve:

# Load required packages
library(PhotoGEA)
library(lattice)

# Define a vector of paths to the files we wish to load; in this case, we are
# loading example files included with the PhotoGEA package
file_paths <- c(
  PhotoGEA_example_file_path('c3_aci_1.xlsx'),
  PhotoGEA_example_file_path('c3_aci_2.xlsx')
)

# Load the data from each file
licor_exdf_list <- lapply(file_paths, function(fpath) {
  read_gasex_file(fpath, 'time')
})

# Get the names of all columns that are present in all of the Licor files
columns_to_keep <- do.call(identify_common_columns, licor_exdf_list)

# Extract just these columns
licor_exdf_list <- lapply(licor_exdf_list, function(x) {
  x[ , columns_to_keep, TRUE]
})

# Combine the data from all the files
licor_data <- do.call(rbind, licor_exdf_list)

# Define a new column that uniquely identifies each curve
licor_data[, 'curve_id'] <-
  paste(licor_data[, 'species'], '-', licor_data[, 'plot'] )

# Organize the data
licor_data <- organize_response_curve_data(
    licor_data,
    'curve_id',
    c(9, 10, 16),
    'CO2_r_sp'
)

# Specify separate mesophyll conductance values for each species; here we use
# arbitrary values since this is just an example
licor_data <- set_variable(
  licor_data, 'gmc', 'mol m^(-2) s^(-1) bar^(-1)',
  id_column = 'species',
  value_table = list(soybean = 0.9, tobacco = 1.1)
)

# Calculate the total pressure
licor_data <- calculate_total_pressure(licor_data)

# Calculate Cc
licor_data <- apply_gm(licor_data)

# Calculate temperature-dependent values of C3 photosynthetic parameters
licor_data <- calculate_arrhenius(licor_data, c3_arrhenius_bernacchi)

# The default optimizer uses randomness, so we will set a seed to ensure the
# results from this fit are always identical
set.seed(1234)

# Fit all curves in the data set
aci_results <- consolidate(by(
  licor_data,
  licor_data[, 'curve_id'],
  fit_c3_aci,
  Ca_atmospheric = 420
))

When this document was generated, evaluating this code required the following amount of time:

#>    user  system elapsed 
#>  13.049   0.039  13.089

The timing results may vary depending on the particular machine used to run the code. Nevertheless, this is a small time investment for an advanced algorithm that uses derivative-free optimizers for robust fitting and calculates nonparametric confidence intervals to determine which estimated parameter values are reliable.

This example contains 13 commands, so it certainly isn’t short; however, a close look reveals that much of the commands are general and would apply to any set of C₃ response curves. In fact, only a few parts would need to be modified, such as the list of files to read, the curve identifier, and the value of mesophyll conductance. While using PhotoGEA, you are encouraged to copy this example and any others to use as the base of your own scripts; work smarter, not harder!

Viewing the Results

Having fit the response curves, it is also possible to view the fits, the parameter estimates, and their confidence intervals. PhotoGEA provides several tools for doing this, which enable users to check the fit quality and ensure that only reliable parameter estimates are used in subsequent analysis.

We can plot the measured values of net assimilation (black circles), the fitted values of net assimilation (An), and each of the limiting assimilation rates calculated during the fitting procedure: the Rubisco limited rate (Ac), the RuBP regeneration limited rate (Aj), and the triose phosphate utilization (TPU) limited rate (Ap). This is a basic quality check where we can make sure that the fits make sense and look believable:

plot_c3_aci_fit(aci_results, 'curve_id', 'Ci', ylim = c(-10, 80))

In this figure, some curves are missing one or more of the potential limiting rates. When this occurs, it means that no points in the curve were found to be limited by that process.

Another way to check the overall quality of the fits is to plot the residuals, which should be randomly distributed:

xyplot(
  A_residuals ~ Ci | curve_id,
  data = aci_results$fits$main_data,
  type = 'b',
  pch = 16,
  grid = TRUE,
  xlab = paste0('Intercellular CO2 concentration (', aci_results$fits$units$Ci, ')'),
  ylab = paste0('Assimilation rate residual (measured - fitted)\n(', aci_results$fits$units$A, ')'),
)

For individual parameters, we can take a look at the best-fit values and the associated confidence intervals. Here is an example showing values of Tp, the maximum rate of triose phosphate utilization.

aci_results$parameters[, c('curve_id', 'Tp_lower', 'Tp', 'Tp_upper')]
#>       curve_id  Tp_lower       Tp Tp_upper
#> 1  soybean - 1 10.068918 10.75812 11.39151
#> 2 soybean - 5a      -Inf       NA      Inf
#> 3 soybean - 5b      -Inf       NA      Inf
#> 4  tobacco - 1      -Inf       NA      Inf
#> 5  tobacco - 2  3.654579       NA      Inf
#> 6  tobacco - 4 12.959903 13.55359 14.14744

Some of these estimates have an upper limit of Inf and a best estimate of NA. A comparison with the fits shown above indicates that for these curves, insufficiently many points were found to be TPU-limited, preventing a reliable estimate of Tp.

It is also possible to plot the best-fit values of a parameter averaged across subsets of the data in a bar chart, where the error bars represent the standard error of the mean. Any values of NA will be excluded. Here we plot values of Vcmax at 25 degrees C for each species.

barchart_with_errorbars(
  aci_results$parameters[, 'Vcmax_at_25'],
  aci_results$parameters[, 'species'],
  xlab = 'Species',
  ylab = paste0('Vcmax at 25 degrees C (', aci_results$parameters$units$Vcmax_at_25, ')'),
  ylim = c(0, 200)
)

Learning More

The PhotoGEA package includes extensive documentation, and more articles are being added all the time: