Primers used in this study.

<p>Fw, forward primers; Rv, reverse primers.</p

Behavior of infrared absorption bands in cell wall isolates.

<p>Cell wall isolates of wild-type and <i>35S::OsEXPA8</i> transgenic line 1 suspension cells on day 10 was used for wall composition analysis. Average and area-normalized Fourier-transform infrared (FTIR) spectra of isolated cell walls from wild-type (lower) and <i>35S::OsEXPA8</i> (upper) line 1 suspension cells.</p

Cell wall stiffness profiles of wild-type and <i>35S</i>::<i>OsEXPA8</i> suspension cells with the passage of time in culture.

<p>(A) and (B) on day 5, (C) and (D) on day 10, (E) and (F) on day 13. Each graph corresponds to stiffness values of 150 FD curves obtained on 30 cells, which derived from three biological replications from <i>35S::OsEXPA</i>8 transgenic line 1. WT: wild-type.</p

Morphological changes in <i>35S</i>::<i>OsEXPA8</i> transgenic lines.

<p>(A) Seven-day-old rice seedlings of wild-type and <i>35S::OsEXPA8</i> transgenic line1 plants. (B, C) Stereoscope images of the primary root of wild-type (B) and <i>35S::OsEXPA8</i> line1 (C) seedlings. Scale bars = 40 µm. (D, E) Epidermal morphology of the root hair growth zones of seminal roots of wild-type (D) and <i>35S::OsEXPA8</i> line1 (E) seedlings. Scale bars = 10 µm. Seedlings were grown for 7 days on half-strength solid MS medium vertically, and the root apex of was observed by stereoscope microscopes and cryo-scanning electron microscope (Cryo-SEM), respectively. (F) Root system architecture of two-month-old wild-type and <i>35S::OsEXPA8</i> line1 plants. (G) The plant morphology of two-month-old wild-type and <i>35S::OsEXPA8</i> line1 plants. (H) Flag leaf phenotype of two-month-old wild-type and <i>35S::OsEXPA8</i> line1 plants.</p

Overexpression of <i>OsEXPA8</i>, a Root-Specific Gene, Improves Rice Growth and Root System Architecture by Facilitating Cell Extension

<div><p>Expansins are unique plant cell wall proteins that are involved in cell wall modifications underlying many plant developmental processes. In this work, we investigated the possible biological role of the root-specific α-expansin gene <i>OsEXPA8</i> in rice growth and development by generating transgenic plants. Overexpression of <i>OsEXPA8</i> in rice plants yielded pleiotropic phenotypes of improved root system architecture (longer primary roots, more lateral roots and root hairs), increased plant height, enhanced leaf number and enlarged leaf size. Further study indicated that the average cell length in both leaf and root vascular bundles was enhanced, and the cell growth in suspension cultures was increased, which revealed the cellular basis for <i>OsEXPA8</i>-mediated rice plant growth acceleration. Expansins are thought to be a key factor required for cell enlargement and wall loosening. Atomic force microscopy (AFM) technology revealed that average wall stiffness values for <i>35S</i>::<i>OsEXPA8</i> transgenic suspension-cultured cells decreased over six-fold compared to wild-type counterparts during different growth phases. Moreover, a prominent change in the wall polymer composition of suspension cells was observed, and Fourier-transform infrared (FTIR) spectra revealed a relative increase in the ratios of the polysaccharide/lignin content in cell wall compositions of <i>OsEXPA8</i> overexpressors. These results support a role for expansins in cell expansion and plant growth.</p></div

Effect of overexpression of <i>OsEXPA8</i> on the root system architecture and plant growth.

<p>(A) The length of primary roots of seven-day-old rice seedlings. (B) The number of lateral roots of seven-day-old rice seedlings. (C) The plant height of two -month-old rice plants. (D) The leaf number per plant of two-month-old rice plants. (E) The length of the flag leaf of two-month-old rice plants. (F) The width of the flag leaf of two-month-old rice plants. Three independent transgenic line 1 (L1), line 4 (L4) and line 5 (L5) were analyzed. WT: wild-type. Values are the means of ten biological replications ± standard error. One independent plant was considered as one biological replication. Asterisks (*) indicate parameters of <i>35S::OsEXPA8</i> transgenic plants were significantly different from that of wild-type plants by statistical analysis using the Student’s <i>t</i>-test program (<i>P</i><0.01).</p

Morphological changes of vascular bundle cells in both leaves and roots in rice <i>35S</i>::<i>OsEXPA8</i> transgenic line 1 plants

<p>. Plants of two-month-old were subject to anatomic analysis. The cells were analyzed in an optical microscope. (A), (C), (E) and (G) wild-type plants; (B), (D), (F) and (H) <i>35S::OsEXPA8</i> transgenic lines; (A) and (B) transverse sections of the flag leaf, (C) and (D) longitudinal sections of the flag leaf; (E) and (F) transverse sections of the lateral root, (G) and (H) longitudinal sections of the lateral root. Scale bars: 25 µm.</p

The growth rate of <i>35S::OsEXPA8</i> suspension cells.

<p>(A) pH value of growth medium, (B) The growth rate of suspension cells per day for <i>35S:OsEXPA</i>8 transgenic line 1 (L1) and wild-type cultures monitored with the passage of time in culture. WT: wild-type. Values are the means of three biological replications ± standard error.</p

Stiffness-properties imaging by atomic force microscopy (AFM) technology.

<p>(A) Optical image of a typical single rice cell sample. The shadow of the AFM cantilever is visible on the left-hand side of the image. (B) A typical force-distance (FD) curve recorded on a single rice cell sample. The upper line represents the FD curve when the tip indents (or penetrates) the cell wall and the lower line represents the FD curve when the tip retracts from the cell wall.</p