EPR spectroscopy and its use in planta—a promising technique to disentangle the origin of specific ROS

Cell extension in the growing zone of plant roots typically takes place with a maximum local growth rate of 50% length increase per hour. The biochemical mechanism of this dramatic growth process is still poorly understood. Here we test the hypothesis that the wall-loosening reaction controlling root elongation is effected by the production of reactive oxygen intermediates, initiated by a NAD(P)H oxidase-catalyzed formation of superoxide radicals (O(2)(.-)) at the plasma membrane and culminating in the generation of polysaccharide-cleaving hydroxyl radicals ((.)OH) by cell wall peroxidase. The following results were obtained using primary roots of maize (Zea mays) seedlings as experimental material. (1) Production of O(2)(.-), H(2)O(2), and (.)OH can be demonstrated in the growing zone using specific histochemical assays and electron paramagnetic resonance spectroscopy. (2) Auxin-induced inhibition of growth is accompanied by a reduction of O(2)(.-) production. (3) Experimental generation of (.)OH in the cell walls with the Fenton reaction causes wall loosening (cell wall creep), specifically in the growing zone. Alternatively, wall loosening can be induced by (.)OH produced by endogenous cell wall peroxidase in the presence of NADH and H(2)O(2). (4) Inhibition of endogenous (.)OH formation by O(2)(.-) or (.)OH scavengers, or inhibitors of NAD(P)H oxidase or peroxidase activity, suppress elongation growth. These results show that juvenile root cells transiently express the ability to generate (.)OH, and to respond to (.)OH by wall loosening, in passing through the growing zone. Moreover, inhibitor studies indicate that (.)OH formation is essential for normal root growth.

The production of reactive oxygen species (ROS) in different plant subcellular compartments is the hallmark of the response to many stress stimuli and developmental cues. The past two decades have seen a transition from regarding ROS as exclusively cytotoxic agents to being considered as reactive compounds which participate in elaborate signaling networks connecting various aspects of plant life. We have now arrived at a stage where it has become increasingly difficult to disregard the communication between different types and pools of ROS. Production of ROS in the extracellular space, the apoplast, can influence their generation in the chloroplast and both can regulate nuclear gene expression. In spite of existing information on these signaling events, we can still barely grasp the mechanisms of ROS signaling and communication between the organelles. In this review, we summarize evidence that supports the mutual influence of extracellular and chloroplastic ROS production on nuclear gene regulation and how this interaction might occur. We also reflect on how, and via which routes signals might reach the nucleus where they are ultimately integrated for transcriptional reprogramming. New ideas and approaches will be needed in the future to address the pressing questions of how ROS as signaling molecules can participate in the coordination of stress adaptation and development and how they are involved in the chatter of the organelles.

• Gas spaces (aerenchyma) form as an adaptation to submergence to facilitate gas exchange. In rice (Oryza sativa), aerenchyma develop by cell death and lysis, which are poorly understood at the cellular level. • Aerenchyma formation was studied in rice stems by light microscopy. It was analyzed in response to submergence, ethylene and hydrogen peroxide (H(2)O(2)) treatment, and in the MT2b::Tos17 mutant. O(2)·(-) was detected with nitroblue tetrazolium and an epinephrine assay. H(2)O(2) was detected with 3,3'-diaminobenzidine. • Aerenchyma develop constitutively in all internodes of the deep-water rice variety Pin Gaew 56, but are absent from the nodes. Constitutive aerenchyma formation was also observed in two lowland rice varieties, albeit to a lesser degree. A larger number of aerenchyma are present in older internodes, and at the top of each internode, revealing developmental gradients. Submergence or treatment with the ethylene-releasing compound ethephon promoted aerenchyma formation in all genotypes analyzed. Pre-aerenchymal cells contain less starch, no chloroplasts, thinner cell walls and produce elevated levels of O(2)·(-) and H(2)O(2) compared with other parenchymal cells. Ethephon promotes O(2)·(-) formation and H(2)O(2) promotes aerenchyma formation in a dose-dependent manner. Further-more, genetic downregulation of the H(2)O(2) scavenger MT2b enhances aerenchyma formation. • Aerenchyma formation is mediated by reactive oxygen species. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).