Volume 4, Issue 2, Pages 331-345 (March 2011) Perturbation of Wood Cellulose Synthesis Causes Pleiotropic Effects in Transgenic Aspen  Joshi Chandrashekhar P. , Thammannagowda Shivegowda , Fujino Takeshi , Gou Ji-Qing , Avci Utku , Haigler Candace H. , McDonnell Lisa M. , Mansfield Shawn D. , Mengesha Bemnet , Carpita Nicholas C. , Harris Darby , DeBolt Seth , Peter Gary F.   Molecular Plant  Volume 4, Issue 2, Pages 331-345 (March 2011) DOI: 10.1093/mp/ssq081 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 PtdCesA8 Transgenes’ Copy Number in 11 Randomly Selected Transgenic Aspen Lines and a Control Line as Detected by Quantitative Real-Time PCR (A) and Semi-Quantitative PCR (B). (A) Copy number estimate of inserted transgenes in the aspen genome (35S:PtdCesA8) was determined by quantitative real-time PCR. (B) Results of semi-quantitative PCR. ‘35S:PtdCesA8’ fragment indicates a 260-bp DNA fragment amplified from the end of 35S promoter to the 5’-end of PtdCesA8 gene using appropriate primers as listed in materials and methods. While ‘TERT’ and ‘POPR’ indicate the two control genes, telomerase with one copy and POPTRCesA1 with two copies, respectively. Note that only one transgene copy insertion in transgenic lines C1, C4, C6, C7, C8, C10, and C11, while two copies are present in C2, C3, C5, and C9. Only a single copy insert containing lines were used for further analysis. 271 WT is the untransformed control. Molecular Plant 2011 4, 331-345DOI: (10.1093/mp/ssq081) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 Morphological Abnormalities Exhibited by Transgenic Aspen Plants. (A) Two dwarf transgenic aspen plants are shown on the right and two untransformed aspen plants of the same age are shown on the left. (B) A close-up of a weak and wavy transgenic aspen stem. (C) Top five leaves of the control (left) and transgenic (right) aspen plants. (D) Root systems of control (left) and transgenic (right) aspen. (E) Transgenic aspen with extensive side branches. (F) Root suckers developed from the control (left) and transgenic (right) aspen plants. Molecular Plant 2011 4, 331-345DOI: (10.1093/mp/ssq081) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 Semi-Quantitative RT–PCR Products from Two Control Aspen Plants (C1 and C2) and Two Selected Transgenic Aspen Lines (TL1 and TL2). Panels (A)–(E) show amplification products using primer pairs specific to genes indicated on the right. These primer pairs are given in the Methods section. Note the loss of amplification products indicative of loss of stable transcripts in TL1 and TL2 lines in the case of PtdCesA8 gene. Molecular Plant 2011 4, 331-345DOI: (10.1093/mp/ssq081) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 Light Microscopy of Control and Transgenic Aspen Lines. Sections of the control Line C1 (A, D, G), transgenic Line TL1 (B, E, H), and transgenic Line TL2 (C, F, I) were stained with Toluidine blue and viewed with DIC optics (A–F), or left unstained and viewed with POL optics (G–I). Rounded vessels in the control (D) contrast with irregularly shaped vessels (marked by black asterisks in (d) and (f)) that often appeared in transgenic secondary xylem (B, C, E, F). In (D, E, F), black arrows are superimposed on one ray in the section, with the tilted arrows showing bends in the ray in (F). Analysis of birefringence in micrographs taken under identical optical conditions and exposure time showed that control Line C1 had the highest ordered cellulose content, and virtually all cells were surrounded by a birefringent boundary (G). In contrast, for transgenic secondary xylem, birefringence was often detected in separate lines or spots, resulting in many cells having an incomplete birefringent boundary (H, I). In (G, H, I), white arrows indicate the overall orientation of the ray system relative to the optical axis. In (H), the relatively straight ray shows that the tissue was not highly contorted, but nearby vessels (marked by asterisks) still lack substantial birefringence in their walls. Bars: in Figure 4A for (A–C), 50 μm; in Figure 4D for (D–F), 10 μm; in Figure 4G for (G–I), 20 μm. Molecular Plant 2011 4, 331-345DOI: (10.1093/mp/ssq081) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 X-Ray MicroCT Imaging and Analysis of Cell Wall and Airspace Size in Control (A–C), TL1 (D–F), and TL2 (G–I). Thresholded images were analyzed for the wall thickness (A, D, G) and airspace (B, E, H) and the distribution of volume is shown in (C, F, I). Molecular Plant 2011 4, 331-345DOI: (10.1093/mp/ssq081) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 Transmission Electron Microscopy of Control and Transgenic Aspen Lines. Transmission electron micrographs of control Line C1 (A), transgenic Line TL1 (B), and transgenic Line TL2 (C). The smooth inner surfaces of control cell walls (A) contrast with more irregular surfaces, including staining irregularities (examples marked by asterisks), in the transgenics (B, C). An inner electron translucent secondary cell wall layer observed in some cells in control Line C1 (a, arrow) was not observed in the transgenic lines. V, highly enlarged vessel element; F, an example of a fiber, of which there are several in each micrograph; R, ray cell with cytoplasmic contents (cy), indicative of its living state before fixation. Bar = 2 μm. Molecular Plant 2011 4, 331-345DOI: (10.1093/mp/ssq081) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 7 Transmission Electron Micrographs of Control Line C1 (A, D), Transgenic Line TL1 (B, F), and Transgenic Line TL2 (C, E). Specimens and images were processed equivalently, so staining qualities can be compared between samples. As already described (Figure 6), staining irregularities in the cell wall (asterisks in (B) and (C)) are characteristic of transgenic lines. In the control (A), residual cytoplasm is marked by (cy) to distinguish it from a cell wall staining irregularity. Sometimes in the transgenic lines (C), cell corners (cc) were depleted of electron dense material. The pits in control (D) and transgenic lines were similar (E, F), having borders (asterisks) and hydrolyzed pit membranes (arrows) in all cases. However, the cell wall irregularities in the transgenics were also observed near pits (E, F). Bar = 0.5 μm for (A–C) and 2 μm for (D, E). Molecular Plant 2011 4, 331-345DOI: (10.1093/mp/ssq081) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 8 X-Ray Diffraction Analysis Was Used to Generate a Relative Crystallinity Value for Transgenic Aspen Mutant versus Wild-Type Aspen Stems. (A) Bragg-Brentano geometries (symmetrical reflection) of transgenic mutant versus wild-type (WT) stem showed that the mutant secondary xylem diffractogram peaks were collapsed and (B) had a 46% lower RCI value than that measured for wild-type wood. Molecular Plant 2011 4, 331-345DOI: (10.1093/mp/ssq081) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions