WP1. Extract pollen grains and macrofossils for a few species of interest from a range of available samples spanning c. 25 thousand years of vegetation history across several radiocarbon-dated lakes, soils, and archaeological sites in the UK, EU, Norway and Russia. Species of interest are grains available as early as the Neolithic (wheat, barley) and key dominant tree species (oak, birch, pine, larch).
WP2. Extract DNA and obtain sequences of Rubisco encoding chloroplast gene rbcL from single pollen grains/macrofossils using the ‘multiplexed inter‐simple sequence repeat (ISSR) genotyping by sequencing’ or ‘MIG‐seq’ method  for the construction of HTS libraries and the genotyping of genome‐wide single nucleotide polymorphisms from low‐quantity DNA templates. Compare sequences with modern ones from the same species.
WP3. If mutations leading to amino acid substitutions are found within the protein coding gene rbcL, assess their impact on enzyme kinetics by both computational biology methods and by introducing selected mutations using CRISPR in plant cell cultures. Purify resurrected “ancient” Rubisco from plant cell cultures and perform biochemical assays to compare Rubisco kinetics at the range of temperatures with modern ones from the same species.
WP4. Modelling exercise to test how future-proof are Rubiscos from studied key species using methodology from  and newly obtained data on enzyme kinetics from both resurrected and modern Rubiscos.
External collaborators outside of IAPETUS: Prof Inger Alsos (The Arctic University, Tromso, Norway), Dr Vladimir Semerikov (Institute of Plant and Animal Ecology, Ekaterinburg, Russia), who will provide access to sediment and soil samples that have previously yielded abundant DNA material [8,9].