Is Chilling Stress in Arabidopsis Caused by ROS Overload in Roots?

Although normally considered as chilling-tolerant, Arabidopsis plants exposed to 2 days of chilling at 4 C show elevated levels of reactive oxygen species (ROS) and inhibited root growth for up to 4 days when transferred back to optimal growing conditions. During this recovery period, ROS levels decline in root tips and in leaves. If plants are pretreated with glycine betaine (GB) prior to the chilling treatment, ROS levels do not increase during chilling and optimal growth begins as soon as plants are transferred back to normal growing conditions without a recovery period. Using microarray technologies, we can show that GB up-regulates several genes in both roots and leaves that reinforce intracellular processes protecting cells from oxidative damage and others that appear to be involved in setting up a scavenging system for reactive oxygen species in cell walls. In roots, GB-activates genes for transcription factors, membrane trafficking proteins (RabA4c, RabB1b), cell wall peroxidases (ATP3a, ATP15a), superoxide dismutases in the cytoplasm, plastids and mitochondria, a mitochondrial catalase, the root specific NADPH-dependent ferric reductase (FRO2) localized to the plasma membrane as well as glutathione and ascorbate metabolizing enzymes in the cytoplasm and cell wall. Genes activated in leaves include transcription factors, several intracellular ROS metabolism enzymes as well as membrane trafficking components. In addition, specific extracellular peroxidases are activated by GB in leaves as well as a plasmamembrane NADPH-dependent ferric reductase (FRO6). Experiments with knockout mutants provide direct evidence that two of the GB-activated genes, one gene coding for a membrane trafficking protein (RabA4c) and the other coding for a putative bZIP transcription factor, are required for GB?s effects on recovery from chilling and ROS accumulation during chilling. GB does not prevent chilling stress in these knockouts. Experiments with RabA4c promoter-GUS and -YFP transgenics show that RabA4c expression coincides with regions of most active ROS accumulation in vascular tissues during chilling stress. Taken together, these results plus the fact that application of ROS directly to plants in the absence of chilling can cause root growth inhibitions suggest that ROS may be the cause of chilling stress.