IC4R001-Phenomics-2010-X6315815

Project Title

A phenomics approach detected differential epigenetic growth regulation between inbreds and their hybrid in Oryza sativa

The Background of This Project

• Inter-specific hybridization can induce instability in both the genome and gene expression, often resulting from changes in epigenetic modification of chromatin (Comai et al. 2000; Liu and Wendel 2003; Shaked et al. 2001). However, inter-specific hybridization is often accompanied by polyploidization and therefore it is difficult to distinguish the effects of the observed epigenetic changes of hybridization from those associated with polyploidization. Although approaches aimed at detecting differentially expressed genes upon hybridization have identified many candidate genes, including those involved in chromatin regulation (Ni et al. 2009; Swanson-Wagner et al. 2006; Wei et al. 2009), most of the changes detected could simply be consequences of the superior characteristics of F1 hybrids, and the upstream signals involved in inducing the phenotypic changes in F1 are still unknown.
• To obtain growth data of seedlings suitable for fine dissection into separate growth parameters, in this study, the researchers grew rice (Oryza sativa L.) lines, including an F1 hybrid produced by crossing a representative japonica cultivar (Nipponbare, NB) to an indica cultivar (Kasalath, KS), in a controlled environment and analyzed their growth by using image-based analysis in which time-lapse images of growing seedlings are taken every 60 min

Plant Culture & Treatment

• Seeds were surface-sterilized as described (Ishizuka et al. 2005). After washing three times with sterile water, seeds were soaked in sterile water with or without TSA (Sigma–Aldrich, St. Louis, MO, USA) and incubated at 30°C for 24 h. Incubated seeds were washed three times with sterile water and grown as described by Ishizuka et al. (2005) in test tubes (30 mm width 9 300 mm height) at 28°C under continuous light (5 mW cm -2 ) for up to 9 days.
• Images of plants were recorded at 60-min intervals using an image capturing system (Tanabata et al. 2008), and leaf length was measured as described previously (Ishizuka et al. 2005). Calculation of the coefficients of the logistic equations used the least squares method with repeated correction after converting the original logistic equation to a logarithmic formula. Individuals growing irregularly and/or giving a coefficient of determination (r 2 ) less than 0.85 in the regression were excluded from further analysis.
  
  

Figure. 1 Characterization of growth parameters of second and third leaves grown in a controlled environment. a Growth curves of the second leaves plotted as averages with standard errors. b Growth parameters of the second leaves. Averages with standard errors of the final length (top), maximum growth rate (middle), and efficiency index (bottom) are shown. Statistically significant differences among the samples are indicated (*** P \ 0.01; n.s., not significant). c Growth curves of the third leaves plotted as averages with standard errors. d Maximum growth rate of the third leaves. Averages with standard errors are shown. Statistically significant differences among the samples are indicated (*** P \ 0.01; ** P \ 0.05)

Research Findings

• The final length of the second leaf was much longer in KS than NB, but similar for KS and F1, and therefore genetic factors determining the second leaf length in KS are inherited dominantly to F1 under our growth conditions (Fig. 1a and Electronic Supplementary Material Table 1). The maximum growth rate of the second leaf was determined using two independent methods: (1) linear regression to a 1,500-min period in the linear growth phase of the second leaf (Fig. 1a); and (2) growth rate profiling, in which the growth rates in 600-min periods for individual seedlings are plotted every 60 min (Sup- plementary Fig. 1). The maximum growth rates determined by these two methods are consistent, and KS and F1 give similar rates while NB shows a distinctly lower rate (Fig. 1b and Supplementary Table 2). The results indicate that genetic factors determining the maximum growth rate of the second leaf are also inherited dominantly from KS to F1.
• The final length of the second leaves of both inbreds and the hybrid decreased as the concentration of TSA increased (Fig. 2a; Table 1), suggesting that the length of the second leaf in both the inbreds and the hybrid depends similarly on HDAC activities. The magnitude of the reduction was small in NB, reflecting its low growth rate in the absence of TSA. The maximum growth rate behaved in a similar way (Fig. 2b; Table 1). In contrast, TSA treatment affected the efficiency index of the second leaf differentially between the inbreds and the hybrid, irrespective of the original growth rates in the absence of TSA (Fig. 2c; Table 1). The two inbreds exhibited a significant dose-dependent decrease in the efficiency index, whereas the hybrid showed no such reduction, indicating that mechanisms regulating the intrinsic rate of growth of the second leaf depend more on HDAC activity in the inbreds than in the hybrid.

Figure. 2 Effect of TSA on the growth parameters of inbreds and their F1 hybrid. Final length (a), maximum growth rate (b), and efficiency index (c) of the second leaf are shown for NB (open circles), KS (open triangles), and F1 (filled squares). d Maximum growth rate of the third leaf

Labs working on this Project

• Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba 305-8602, Ibaraki, Japan

Corresponding Author

Yoshiki Habu(email: habu@affrc.go.jp)