Limited ventilation causes stress and changes in Arabidopsis morphological, physiological and molecular phenotype during in vitro growth

Abstract

A huge number of experiments in plant biology are conducted in sterile, sealed containers, providing environmental stability and full control of factors influencing the plant system. With respect to roots the in vitro growth has another benefit - the ease of conducting visual observations when grown in transparent media. Moreover, straightforward measurements of in vitro grown root systems make them a sensitive and convenient sensor of multiple stresses which may occur during experiments. In order to optimize root nematode infection tests for Arabidopsis mutants with relatively mild phenotypes, two Petri dish sealing techniques were tested (permeable medical adhesive tape and a popular non-permeable plastic film). Using standard experimental settings applied for infection tests, the root architecture, nematode infections, ion leakage, efficiency of photosynthesis, ethylene (ET) production, and CO₂ accumulation were monitored in Arabidopsis thaliana Ws-0 wild-type and lsd1 (lesion stimulating disease 1) plants, which is a conditional dependent programmed cell death mutant. All tested parameters gave statistically significant differences between the analyzed sealing tapes, indicating the importance of air exchange. This factor is quite obvious but often ignored in experiments performed in Petri dishes. The results clearly indicate that stress is greater in air-tight sealed plates. These observations were supported by the great expression variation of several marker genes associated with reactive oxygen species (ROS), ET, salicylic (SA), and jasmonic acid (JA) biosynthesis and signaling in two-week-old seedlings. These results are discussed in light of the observed changes in the ET and CO₂ concentration. Our results clearly indicate the importance of culture parameters for monitoring of abiotic and biotic stress responses in laboratory conditions, including accurate mutant phenotyping.

Keywords: Arabidopsis thaliana; Culture vessel ventilation; In vitro; Root growth; Sealing tape.