Fluctuating light experiments and semi-automated plant phenotyping enabled by self-built growth racks and simple upgrades to the IMAGING-PAM

Abstract

BackgroundOver the last years, several plant science labs have started to employ fluctuating growth light conditions to simulate natural light regimes more closely. Many plant mutants reveal quantifiable effects under fluctuating light despite being indistinguishable from wild-type plants under standard constant light. Moreover, many subtle plant phenotypes become intensified and thus can be studied in more detail. This observation has caused a paradigm shift within the photosynthesis research community and an increasing number of scientists are interested in using fluctuating light growth conditions. However, high installation costs for commercial controllable LED setups as well as costly phenotyping equipment can make it hard for small academic groups to compete in this emerging field.ResultsWe show a simple do-it-yourself approach to enable fluctuating light growth experiments. Our results using previously published fluctuating light sensitive mutants, stn7 and pgr5, confirm that our low-cost setup yields similar results as top-prized commercial growth regimes. Moreover, we show how we increased the throughput of our Walz IMAGING-PAM, also found in many other departments around the world. We have designed a Python and R-based open source toolkit that allows for semi-automated sample segmentation and data analysis thereby reducing the processing bottleneck of large experimental datasets. We provide detailed instructions on how to build and functionally test each setup.ConclusionsWith material costs well below USD$1000, it is possible to setup a fluctuating light rack including a constant light control shelf for comparison. This allows more scientists to perform experiments closer to natural light conditions and contribute to an emerging research field. A small addition to the IMAGING-PAM hardware not only increases sample throughput but also enables larger-scale plant phenotyping with automated data analysis.

Comparative analysis of thylakoid protein complexes in state transition mutants nsi and stn7: focus on PSI and LHCII

“…For lpBN gel electrophoresis and subsequent MS analysis, Arabidopsis thaliana wt plants (Col-0) and the T-DNA mutant lines nsi-1 (SALK_033944), nsi-2 (SALK_020577) (Koskela et al 2018) and stn7 (SALK_073254) (Tikkanen et al 2006) were grown in 8 h light/16 h darkness at photosynthetic photon flux density (PPFD) of 100 µmol m −2 s −1 , 50% humidity and + 23 °C. For physiological experiments, plants were grown in 12 h light/12 h darkness at PPFD of 90 µmol photons m −2 s −1 for 2 weeks followed by a shift to fluctuating light (cycles of 4 min 90 µmol m −2 s −1 followed by 1 min 900 µmol m −2 s −1 ) for 2 weeks (Schneider et al 2019).…”

“…Plants were dark-adapted for 20 min before the induction curve was measured for 300 s at 186 µmol m −2 s − 1 with saturating pulses given every 20 s. The reported Y(II), NPQ and Y(NO) values were calculated by averaging the last two data points of the induction curve. Data analysis of the 15-day light-treatment experiment were carried out using the ImagingPAMProcessing toolkit (Schneider et al 2019).…”

The Arabidopsis T‐DNA mutant SALK_008491 carries a 14‐kb deletion on chromosome 3 that provides rare insights into the plant response to dynamic light stress

“…In our initial phenotyping experiment (Figure 1), SALK_008491 plants showed decreased growth rates in response to fluctuating light. To dissect this behavior, 14‐day‐old plants (12/12 h, continuous light 120 μmol photons m −2 s −1 ) were exposed to a long‐term fluctuating light treatment (12/12 h; 1 min 900 μmol photons m −2 s −1 , 4 min 90 μmol photons m −2 s −1 ) (Schneider et al, 2019). After 2 weeks of treatment, we determined photosynthetic parameters and aboveground biomass (Figure 6a–c and Movie S1).…”

“…A. thaliana WT and mutant plants (accession Columbia‐0 [Col‐0]) were germinated on one‐half Murashige and Skoog (MS), 1% phytoagar plates pH 5.8 for 7 days in a growth chamber (CU‐41L4; Percival Scientific) with 150 μmol photons m −2 s −1 of continuous light (cool‐white fluorescent bulbs), 16/8‐h light:dark cycle. After potting plants on soil (Sungro Professional Growing Mix #1, Sun Gro Horticulture, Agawam, MA, USA), they were shifted to self‐built growth racks with 120 μmol photons m −2 s −1 of continuous light in 12/12‐h light:dark cycles and room temperature 22°C (Schneider et al, 2019). When individuals were designated for light treatments, 2‐week‐old plants were moved either into constant high light (900 μmol photons m −2 s −1 ) or into fluctuating light for 2 weeks.…”

“…Subsequently, plants were dark‐adapted for 15 min followed by recording of a standard induction curve at 186‐μmol photons m −2 s −1 actinic light (Kunz et al, 2009). Image extraction and data analysis were performed as described earlier (Schneider et al, 2019).…”