IC4R006-Microarray-2014-24913626

Project Title

Microarray analysis of laser-microdissected tissues indicates the biosynthesis of suberin in the outer part of roots during formation of a barrier to radial oxygen loss in rice (Oryza sativa)

The Background of This Project

• Root growth into hypoxic or anoxic waterlogged substrates relies on internal diffusion of oxygen (Armstrong, 1979). The roots of waterlogging-tolerant species, such as rice (Oryza sativa), typically contain a large volume of aerenchyma. The aerenchyma provides a low-resistance pathway for diffusion of oxygen from the shoot base to the root tip (Armstrong, 1979). The roots of some wetland species, including rice, also possess a barrier to radial oxygen loss (ROL) within the basal zones (Armstrong, 1971; Visser et al., 2000; Colmer, 2003a; Garthwaite et al., 2003; Garthwaite et al., 2008; Abiko et al., 2012), so that ROL occurs predominately from short lateral roots (Armstrong and Armstrong, 2005) and the apical few centimetres of the main axes of adventitious roots (Fig. 1A). A barrier to ROL prevents loss of oxygen from the basal part of roots, which can enhance oxygen transport via the aerenchyma to the root tip. In the roots with an ROL barrier, oxygen at the root tip can be maintained at a higher level to allow root elongation into hypoxic/anoxic soil. The barrier might also impede entry of phytotoxins from chemically reduced water- logged soil.
• Another key feature contributing to waterlogging tolerance is aerenchyma formation. In rice roots, aerenchyma is formed constitutively, but the amount can be enhanced by soil water-logging (Armstrong, 1971; Pradhan et al., 1973; Jackson and Armstrong, 1999), low oxygen (Colmer et al., 2006), and ethylene (Justin and Armstrong, 1991; Colmer et al., 2006). On the other hand, the barrier to ROL is inducible in rice roots, forming in stagnant or waterlogged conditions, but not (or only weakly) in well-drained or aerated conditions (Colmer et al., 1998; Colmer, 2003a; Colmer et al., 2006; Insalud et al., 2006; Kotula et al., 2009; Shiono et al., 2011). Interestingly, low oxygen, ethylene, and elevated CO 2 , which arise from natural waterlogged soil, are not involved in triggering for- mation of the barrier to ROL in roots of rice (Colmer et al., 2006).
• The signal that triggers formation of the barrier to ROL is not known yet, but it may involve root exudates, cellular degradation products (Armstrong and Armstrong, 2001; Voesenek and Sasidharan, 2013) or phytotoxins present in chemically reduced waterlogged soils (e.g. Fe 2+ , sulphide, and microbial metabolites; Armstrong, 1979; Armstrong and Armstrong, 2005; Mongon et al., 2014). Aerenchyma and the barrier to ROL in roots are regarded as key features contrib- uting to long-distance oxygen transport and waterlogging tolerance in many wetland species (Armstrong, 1979; Jackson and Drew, 1984; Justin and Armstrong, 1987; Colmer, 2003b; Colmer and Voesenek, 2009; Nishiuchi et al., 2012). However, the molecular mechanism of ROL barrier formation is poorly understood (Shiono et al., 2008).

Plant Materials & Treatment

• Lowland rice (O. sativa L. cv. Nipponbare) was grown in a nutrient solution of the same composition as used in earlier studies of rice (Colmer, 2003a; Shiono et al., 2011). Plants were supported at the stem base in pots (light-shielding pots so roots were in darkness) within a controlled-environment chamber (24-h light, 28 °C, relative humidity over 50%, photosynthetic photon flux density at 214 μmol m –2 s –1 ). In each experiment, seeds were soaked for 30 min in 0.6% (w/v) sodium hypochlorite for surface sterilization. Seeds were then washed thoroughly with deionized water, and then the seeds were placed in Petri dishes containing about 5 mm deionized water for 2 d in darkness at 30 °C. After 2 d imbibition, germinated seeds were placed on stainless mesh floating on aerated quarter-strength nutrient solution and exposed to light. After 6 d imbibition, each seedling was held with soft sponge floating on aerated full-strength nutrient solution. After 9 d imbibition, seedlings were transferred to 5-l pots (height 250 mm, width 120 mm, breadth 180 mm) containing aerated full-strength nutrient solution. Solutions were renewed every 7 d.
• In each experiment, pots were arranged in a completely randomized design. 23-d-old plants were either continued in aerated solution or transplanted into N 2 -flushed or stagnant deoxygenated solution for 9 h (Fig. 1C). In N 2 -flushed nutrient solution, oxygen level was kept hypoxic (dissolved oxygen <1.0 mg l –1 ). Stagnant solution contained 0.1% (w/v) dissolved agar and was deoxygenated (dissolved oxygen <1.0 mg l –1 ) prior to use by preflushing with N 2 gas. The dilute agar prevents convective movements in solution (‘stagnant’ treatment) so this treatment mimics better than other solution culture methods the changes in gas composition found in waterlogged soils (e.g.

decreased oxygen, increased ethylene) (Wiengweera et al., 1997).

• Adventitious roots of 23-d-old plants were classified as short (65–85 mm) or long (115–135 mm), based on the length of the main axis at commencement of treatments. Selected short and long adventitious roots were marked near the base using small loops of sewing cotton, so these could be recognized over time.

Labs working on this Project

• Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjyojima, Eiheiji, Fukui 910-1195, Japan.
• Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan.
• Graduate School of Agriculture and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
• Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117585, Singapore.
• School of Plant Biology and Institute of Agriculture, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
• National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
• Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1–308 Nonoichimachi, Ishikawa 921-8836, Japan.

Corresponding Author

Katsuhiro Shiono (email: shionok@fpu.ac.jp ) & Mikio Nakazono (email:nakazono@agr.nagoya-u.ac.jp)