Demonstration of osmotically dependent promotion of aerenchyma formation at different levels in the primary roots of rice using a 'sandwich' method and X-ray computed tomography

The effect of environmental factors on the regulation of aerenchyma formation in rice roots has been discussed for a long time, because aerenchyma is constitutively formed under aerated conditions. To elucidate this problem, a unique method has been developed that enables sensitive detection of differences in the development of aerenchyma under two different environmental conditions. The method is tested to determine whether aerenchyma development in rice roots is affected by osmotic stress. To examine aerenchyma formation both with and without mannitol treatment in the same root, germinating rice (Oryza sativa) caryopses were sandwiched between two agar slabs, one of which contained 270 mm of mannitol. The roots were grown touching both slabs and were thereby exposed unilaterally to osmotic stress. As a non-invasive approach, refraction contrast X-ray computed tomography (CT) using a third-generation synchrotron facility, SPring-8 (Super photon ring 8 GeV, Japan Synchrotron Radiation Research Institute), was used to visualize the three-dimensional (3-D) intact structure of aerenchyma and its formation in situ in rice roots. The effects of unilateral mannitol treatment on the development of aerenchyma were quantitatively examined using conventional light microscopy. Structural continuity of aerenchyma was clearly visualized in 3-D in the primary root of rice and in situ using X-ray CT. Light microscopy and X-ray CT showed that the development of aerenchyma was promoted on the mannitol-treated side of the root. Detailed light microscopic analysis of cross-sections cut along the root axis from the tip to the basal region demonstrated that aerenchyma developed significantly closer to the root tip on the mannitol-treated side of the root. Continuity of the aerenchyma along the rice root axis was morphologically demonstrated using X-ray CT. By using this osandwich' method it was shown that mannitol promoted aerenchyma formation in the primary roots of rice.