Glycine betaine (GB) can confer tolerance to several types of stress at low concentrations, either after application to plants or in transgenics engineered to overproduce GB. Based on earlier studies on levels of GB in plants and evidence for effects on gene expression, we hypothesized that at least part of this effect could be ascribed to the activation of the expression of stress tolerance genes. Using a strategy based on high‐throughput gene expression analysis with microarrays followed by confirmation with northern blots, we identified Arabidopsis genes upregulated in roots that reinforce intracellular processes protecting cells from oxidative damage and others that appear to be involved in reinforcing a scavenging system for reactive oxygen species (ROS) in cell walls. Upregulated genes in roots include those for the membrane‐trafficking RabA4c, the root‐specific NADPH‐dependent ferric reductase (FRO2) localized to the plasma membrane, mitochondrial catalase 2 and the cell wall peroxidase ATP3a. Comparative studies with wild‐type Arabidopsis and knockout mutants for the membrane‐trafficking RabA4c gene demonstrated that the mutants respond only slightly to GB, if at all, compared with wild‐type in relation to root growth recovery after chilling stress, demonstrating the role of RabA4c in relation to the GB effect. The results point toward links between oxidative stress, gene expression, membrane trafficking and scavenging of ROS such as superoxide and hydrogen peroxide in relation to GB effects on chilling tolerance in plants.
It was known that application of glycine betaine (GB) to plants could improve tolerance to stress caused by chilling, frost, salt, drought and high light intensities, and that this effect was accompanied by gene expression changes, but whether the gene expression changes were implicated in GB's effect and which genes were involved has been unclear. In the fourth issue of Physiologia Plantarum, we identified genes upregulated by GB that are involved in reactive oxygen species (ROS) metabolism and membrane trafficking processes. Direct evidence was provided for a role for a membrane trafficking protein (RabA4c) in GB's effect on ROS accumulation during chilling. In this Addendum, we discuss our findings that chilling stress is so closely linked with ROS accumulation. Chilling elevates ROS levels and results in inhibited root growth upon transfer of plants back to normal growing conditions. During the 2-4 day recovery period, ROS levels decline in root tips and in leaves. If ROS accumulation in response to chilling is blocked by pretreatment with GB, optimal root growth begins as soon as plants are transferred back to normal growing conditions without a recovery period, suggesting that chilling stress involves a ROS signaling pathway.
FRO2 (At1g01580) codes for an NADPH-dependent ferric reductase in plasma membranes of root epidermal cells with a demonstrated role in iron uptake by plants. Ferric reductase activity has been shown to be the rate-limiting step for iron uptake in strategy I plants like Arabidopsis and in rice, but it has been unclear whether FRO genes have other physiological functions. We hypothesized that FRO2 was involved in chilling stress tolerance because its expression was upregulated by treatment of plants with glycine betaine (GB), a chemical that prevents reactive oxygen species (ROS) signaling in chilling stress. This idea was confirmed by showing that the FRO2 null mutant frd1-1 failed to respond to GB in chilling assays either in relation to root growth recovery or inhibition of ROS accumulation. Measurements of ferric reductase activity in wild-type plants treated with GB before chilling showed no significant GB effect compared with controls. In addition, 35S-FRO2 transgenics with elevated mRNA levels did not have improved chilling tolerance. However, ferric reductase activity in wild-type plants or 35S-FRO2 transgenics pretreated with GB was several-fold higher after chilling compared with non-pretreated controls. These experiments identify a new physiological function for FRO2, i.e. blocking ROS accumulation during chilling. They also suggest that GB has a major effect on FRO2 activity posttranscriptionally in the cold.
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