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

Figure 3. Expression of selected suberin and lignin biosynthesis genes in the outer part of root (OPR) of rice.

• 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.

Figure 2. Numbers of up- and downregulated gene probes at the outer part of root (OPR) during formation of a radial oxygen loss (ROL) barrier in rice (i.e. the OPR along the basal part of long adventitious roots, 9 h treatment in deoxygenated stagnant 0.1% agar nutrient solution).

Research Findings

Figure 4. Expression of selected suberin biosynthesis genes in the outer part of root (OPR), cortical parenchyma (CP), and central cylinder (CC) of roots of rice.

• As a result, the signal intensities of 167 (0.4%) of the 41 880 gene probes spotted on the microarray slide were significantly higher or lower in OPR tissue forming the barrier to ROL than in OPR tissues that were not forming a barrier. Of the 167 gene probes, 125 (0.3% of the probes on the array) were upregulated and 42 (0.1% of the probes on the array) were downregulated (Fig. 2). After excluding homolo- gous gene probes, 98 genes were upregulated and 30 genes were downregulated under conditions inducing ROL barrier formation in the OPR of the basal part of long adventitious roots in stagnant deoxygenated nutrient solution. The most promi- nent GO classifications of the up- and downregulated genes were metabolic and cellular processes.
• To confirm the key microarray results, this work selected 13 genes (11 upregulated genes that were predicted to be involved in suberin biosynthesis and two downregulated genes) for semiquantitative RT-PCR using RNA obtained from the laser microdissection-iso- lated OPR from long adventitious roots grown in stagnant deoxygenated condition. The 11 upregulated genes were predicted to be involved in suberin biosynthesis. One of these 11 genes, a POD gene (LOC_Os06g16350) is involved in the biosynthesis of both suberin and lignin. The two downregulated genes were PHOSPHOENOLPYRUVATE CARBOXYLASE (PEPC) (Lin et al., 2004), which sup- plies oxaloacetate to the TCA cycle and CHAPERONIN 60/TCP-1 FAMILY PROTEIN (CPN60), which is required for correct protein folding. The expression of each of these genes in the OPR in long adventitious roots under stagnant deoxygenated conditions were upregulated or downregulated in agreement with the microarray findings.
• To determine whether upregulation was specific to the OPR, this work selected four genes (WRKY, CYP, GPAT, and ABCG5) from the previously selected 11 upregulated genes. The expression of these genes were compared in three tissues under aerated and stagnant conditions. The three tissues were OPR (including exodermis, and sclerenchyma), cortical parenchyma (CP, excluding exodermis, sclerenchyma, and endodermis), and central cylinder (CC, including endodermis) from cross-sections of long adventitious roots. Under stagnant conditions, each of the four genes was highly expressed in the OPR (Fig. 4). CYP (LOC_Os04g47250) was also expressed in the CP and CC in stagnant conditions. GPAT (LOC_Os05g38350) was also expressed in the CP; however, the mRNA levels in the OPR were greater than those in the CP and CC. All of these selected upregulated genes involved in suberin biosynthesis were more strongly expressed in the OPR than in the CP and CC, indicating a strong association between the expression of these genes in the OPR and ROL barrier formation.

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)