Transgenic poplar generation and OsSWN1 expression
The tissue-preferential pattern of NST3 expression met our objective, i.e., SCW formation was promoted in poplar stem xylem by the chimeric overexpression of OsSWN1 [22]. After the genetic transformation of the hpCAD19 line via an Agrobacterium-mediated transformation procedure, we obtained over 10 independent transgenic lines with the chimeric OsSWN1 construct. We selected four independent OsSWN1-expressing lines (lines #B, #C, #E, and #G) for further analysis based on their growth stability during successive propagation by in vitro cuttings in the culture medium. Although they could be maintained by sequential in vitro propagation for over a year, cuttings from lines #C and #G were relatively difficult to root compared to the wild-type and other transgenic lines (data not shown).
Figure 1A shows each transgenic line placed in two rows in a culture room after soil cultivation for 4 months. Although the height of line #B (60.5 ± 13.7 cm) was slightly lower than that of wild-type plants (79.3 ± 3.9 cm), no statistical differences were detected at P < 0.01 level among the wild-type and hpCAD-19 (85.5 ± 7.8 cm) lines, and the plant lines #B and #E (76.7 ± 3.8 cm). In contrast, growth retardation was observed in lines #C (54.0 ± 7.3 cm) and #G (33.5 ± 17.7 cm). This might be due, in part at least, to the introduction of the chimeric OsSWN1 construct into the plants.
To validate chimeric OsSWN1 expression, semiquantitative RT-PCR was performed using total RNA isolated from each plant. Expression was detected in the four OsSWN1 lines, but the OsSWN1 level in line #E was apparently lower than that of the other OsSWN1 lines (Fig. 2). These results indicated that OsSWN1 expression level varied among the different plant lines though difference in the expression level among the lines #B, #C, and #G was unclear in our present data. No amplified DNA derived from the transgene was observed in the wild-type and hpCAD19 plants.
Endogenous CAD1 expression
As reported previously, heterologous OsSWN1 expression can induce CAD (CAD-D) expression in transgenic Arabidopsis [16] and CAD1 expression in transgenic poplar [25]. These results suggest that simultaneous expression of chimeric OsSWN1 and CAD1-RNAi transgenes is expected to result in a conflicting response on endogenous CAD1 expression in the present lines. Thus, we checked whether sufficient CAD1 suppression could still be achieved in the four transgenic OsSWN1 lines. As indicated in Additional file 1: Figure S1, CAD1 expression remained depressed in all four OsSWN1 lines and no significant differences were detected compared to the hpCAD19 line, although the suppression levels varied slightly among the tested plants.
Unlike wild-type poplar, the CAD-deficient hpCAD19 line exhibited red coloration in its xylem tissue that turned to brown after drying (Fig. 1B). This coloration is a known indicator of CAD deficiency in various plant species [14]. As indicated in Fig. 1B, the four OsSWN1 lines also showed brown xylem tissue coloration, indicating that endogenous CAD1 expression was still efficiently suppressed in the transgenic poplars. This supports the findings of the real-time PCR analysis shown in Additional file 1: Figure S1.
Histochemical analysis of stem tissues
For further characterization of the effect of chimeric OsSWN1 expression on accelerating SCW formation, we analyzed lignin deposition and cell wall thickness in stems using thin sections of stem tissues. At lower magnification (10× for wild type and 20x for other lines) under bright field observation, no apparent histochemical differences were observed between the wild-type, hpCAD19, and OsSWN1-expressing lines (Fig. 3A–F). In contrast, microscopic observation of autofluorescence from plant tissues differed among the tested lines. No autofluorescence occurred in the pith parenchyma tissues of the wild-type (Fig. 3G), hpCAD19 (Fig. 3H), and line #E (Fig. 3K) plants. In contrast, strong autofluorescence was detected in the three OsSWN1 lines (#B, #C, and #G; Fig. 3I, J, L) in which the OsSWN1 expression was apparently higher than the line #E, indicating that lignification was accelerated in the pith tissue, where less lignification generally occurs [29]. Accelerated pith lignification was previously reported in OsSWN1-expressing poplars generated in a wild-type background [16].
Under microscopic observation of safranin-stained sections at high magnification, cell wall thickness of xylem tissue in hpCAD19 plants seemed to be thinner than that of the wild-type plants, though the differences were not remarkable (Fig. 3M, N). In contrast, the cell wall thickness in lines #B and #G (Fig. 3O, R) were higher than those of hpCAD19 and wild-type plants. These results suggested that OsSWN1 expression induced enhanced cell wall formation in transgenic plants.
Quantitative characterization of accelerated cell wall formation by imaging analysis
To confirm the enhanced cell wall thickness in OsSWN1 lines, an image analysis of wood fiber cells was performed based on microscopic imaging of safranin-stained thin sections as described in previous studies [16, 25]. Analysis showed that the average cell wall thickness of fibers in the hpCAD19 line (1.63 ± 0.25 μm, N = 1405) was slightly but significantly lower than that of the wild-type line (1.81 ± 0.27 μm, N = 934) (Fig. 4). This result suggested that lignin modification by CAD1 suppression accompanied reduced cell wall thickness in the fibers. In contrast, the cell wall thickness of the OsSWN1-expressing lines was significantly higher (3.7% higher in #E, 17% higher in #C, and 29% higher in #B and #G) than that of the wild-type line (Fig. 4). Our data indicated that OsSWN1 expression promoted cell wall thickening even under CAD1 suppression and restored the reduced thickness observed in the hpCAD19 line.
Moisture content in fresh wood
Generally, enhanced wall thickness is expected to induce increased wood density. Since the moisture content of fresh wood has a roughly negative correlation with its density, we calculated the moisture content ([fresh weight − dry weight]/fresh weight) of debarked wood prepared from the stems of 4-month-old plants immediately after harvest and subsequent air-drying for 1 month under room conditions. Although wood moisture could not be completely removed by this procedure, moisture content of the whole stem samples could be estimated. As shown in Additional file 1: Figure S2, the moisture content of the wild type and hpCAD19 line were on the same level. In contrast, those in lines #B and #C were significantly reduced by 14% and 15%, respectively, compared to the wild type. Compared to hpCAD19, the moisture content in lines #B, #C, and #G were also significantly reduced by 15%, 16%, and 7%, respectively. In contrast, the moisture content in line #E (in which the OsSWN1 expression was relatively low) was comparable to those of the wild type and the hpCAD19 line. Collectively, our results suggested that chimeric OsSWN1 expression led to decreased fresh moisture content in the debarked stem. The decrease might be due to decrease in intracellular space occurring along with accelerated thickening of SCW in xylem cells as previously reported in other transgenic poplars harboring OsSWN1 [25]. The enhanced SCW deposition also led to increase in wood density as discussed in the next section.
Stem wood density
The wood density of the top 3-cm part of harvested transgenic stems was determined based on Archimedes’ principle (black bars in Fig. 5). The averaged values of three biological samples from lines #B and #C were 19% and 24% higher than that of the wild-type plants, respectively. In contrast, the wood density of hpCAD19 plants was 15% lower than that of the wild-type plants. Although reduced wood density in hpCAD19 plants has not been previously reported, wood density in hpCAD24 line with the same CAD1-RNAi construct was significantly lower than that of its wild-type counterparts [30]. In contrast to the higher level of cell wall thickness enhancement detected in line #G (Fig. 4), no difference in wood density was observed compared to that of the wild-type plant. This may partially be due to the differences in stem parts used in these analyses and the growth retardation of line #G.
Particle density (packed density) was also measured using CWR prepared from stem wood of each line (white bars in Fig. 5). The values were significantly increased in lines #B, #C, and #G which exhibited higher OsSWN1 overexpression compared to wild type, hpCAD19, and line #E with lower expression of OsSWN1. Collectively, present results indicate that OsSWN1 overexpression contributes to increase in the stem wood density of the plants with an hpCAD19 background, as reported previously in a wild-type background [16, 25].
Lignin characterization
Lignin content in CWR, as determined by an acetyl bromide protocol, showed no significant differences between the wild-type and the hpCAD19 lines (Additional file 1: Figure S3). Similarly, the lignin contents of lines #C, #E, and #G did not significantly differ from that of their host plant, line hpCAD19. In contrast, that of line #B was significantly increased by 14% compared to the hpCAD19 line. This discrepancy observed in the OsSWN1 lines might be due to the expression level of the chimeric transcription factor in each line or the position effect of the gene transfer site in the chromosome.
In addition to the lignin content, the monomeric composition of lignin was also determined by thioacidolysis (Additional file 1: Figure S4). Detected levels of total thioacidolysis monomers (G plus and S monomers) in the hpCAD19 line were lower than those of wild-type plants, as reported previously [11]. The syringyl to guaiacyl (S/G) ratio of the hpCAD19 line (1.40) was relatively lower than that of the wild-type plants (1.66, P = 0.074). Compared to the hpCAD19 plants, the level of the sum of the two monomers was increased in the four OsSWN1 expressing lines but no significant differences were detected. In contrast, the G monomer level in lines #B, #C, and #G (which had higher OsSWN1 expression) was significantly higher than that in the hpCAD19 plants. This change led to a significant reduction of the S/G ratio in these three lines (0.79, 0.88, and 0.98 for the #B, #C, and #G lines, respectively) compared to the hpCAD19 line. In line #E, which displayed relatively low OsSWN1 expression, the S/G ratio (1.29) was comparable to that of the hpCAD19 line. These results suggested that chimeric OsSWN1 expression in plants with an hpCAD19 background were more effective in changing lignin composition than lignin content, as observed in previous studies studying the effects of OsSWN1 expression in a wild-type background [25].
As indicated in Additional file 1: Figure S1, remained CAD1 suppression in the four OsSWN1 lines could be confirmed by real-time PCR. The reduction of CAD1 expression in poplar as well as other plant species known to lead incorporation of coniferaldehyde and sinapaldehyde into the lignin [11]. The incorporation could be detected as release of indene derivatives by thioacidolysis of lignin. Additional file 1: Figure S5 shows the indene release from the four OsSWN1 lines as the hpCAD19 line. In contrast, no release from the wild-type plants could be observed. These results indicate that substantial amount of the hydroxycinnamaldehydes remains to be incorporated into lignin under overexpression of chimeric OsSWN1.
Saccharification efficiency
Lignin modification via CAD1 suppression improves delignification efficiency under alkaline conditions [15] and the subsequent saccharification of CWRs [11]. Saccharification efficiency was measured after a simple alkaline pretreatment to examine the effect of OsSWN1 expression-induced enhancement of cell wall thickness.
As in the previous study [11], CWR of the hpCAD19 line exhibited high saccharification efficiency compared to wild-type plants (Fig. 6). Although the saccharification efficiency in the OsSWN1-expression lines was still higher than that of the wild-type line, the efficiencies of the #B, #C, and #G lines were slightly but significantly lower than that of the hpCAD19 line. In contrast, no significant difference was observed between the hpCAD19 and line #E plants. These results indicated that excessive overexpression of chimeric OsSWN1 created a trade-off between enhanced cell wall thickness and improved saccharification efficiency by lignin modification; however, the improved characteristic of easy delignification by CAD1 suppression remained after cell wall thickness was enhanced by chimeric OsSWN1 overexpression.