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Anatomy and Morphology |
The Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340 USA; Wood Materials and Engineering Laboratory, Washington State University, Pullman, Washington 99164-1806 USA
Received for publication May 28, 2006. Accepted for publication April 13, 2007.
ABSTRACT
To our knowledge, xylary reaction tissue has never been reported in a forage crop species. Here we report the discovery of reaction tissue in a transgenic line of Medicago sativa (pC3H, for the gene for p-coumarate-3-hydroxylase) with reduced lignin content and in the wild-type (WT) line. Based on microscopy and biomechanical testing of internodal alfalfa branch sections, the transgenic (pC3H-I) line, relative to the WT (1) apparently formed more reaction tissue containing gelatinous fibers with adjacent thick-walled fibers (presumed to be "intermediate" tissue) more rapidly, (2) had more xylem tissue, and (3) had comparable tensile dynamic modulus properties. These findings thus establish the (limited) ability of this perennial angiosperm to form (inducible) reaction tissue in a manner somewhat analogous to that of woody arborescent angiosperms. The potential of effectuating reductions in lignin amounts in (woody) angiosperms with increased formation of reaction (tension wood) tissue is discussed because reaction tissues are often viewed as a deleterious trait in processing for many agronomic/industrial applications, especially with the current interest in biofuels.
Key Words: alfalfa • gelatinous fiber • lignin • Medicago sativa • p-coumarate-3-hydroxylase (pC3H) • reaction tissue • tensile modulus • tension wood • zinc chloro-iodide
Lignin, nature's second most abundant vascular plant biopolymer after cellulose, has essential roles in providing both structural support and conduits for water and nutrient transport; it also provides a physical barrier to opportunistic pathogens/herbivores (Lewis et al., 1999
). Recently, engineered reduction of lignin in transgenic lines of economically important plants has been studied in several laboratories, with various potential benefits being anticipated (Anterola and Lewis, 2002
). These benefits include decreased generation of lignin byproduct waste during pulp and paper production, improved animal feed digestibility, as well as better lignocellulosic feedstocks for biofuel/bioethanol production. In most cases, however, reductions in lignin content are accompanied by unintended pleiotropic consequences, such as stunted growth (Patten et al., 2005
; Laskar et al., 2006
) and/or weakened vasculature systems and reduced stem strengths (see Anterola and Lewis, 2002
for examples). Such effects remind us that the general understanding of plant physiology and metabolism—particularly regarding the formation of various lignified and unlignified cell wall types—is still severely lacking (for example, see Davin and Lewis, 2005
).
One of the species being examined for reduced lignin production is alfalfa (Medicago sativa L.), a major forage crop of high nutritional value with an important role in soil nitrogen recycling. In this study, therefore, we examined the effects of modulation of p-coumarate-3-hydroxylase (pC3H) gene expression in alfalfa, on lignin deposition and overall stem vasculature properties and architecture. This enzyme was discovered by Heller and Kühnl (1985)
using parsley (Petroselinum crispum) cell suspension cultures, with p-coumaryl shikimate being its substrate and not p-coumaric acid (Fig. 1A, B). The encoding gene in Arabidopsis thaliana was later described by Schoch et al. (2001)
.
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64%) in the downregulated line, material strength properties of young alfalfa branches were similar in both lines. We therefore propose that formation of reaction tissue may increase in alfalfa and other angiosperms when the overall vasculature is weakened through reduced lignin content. Increased formation of reaction tissue might compensate for the loss of lignin and might help maintain the vasculature's biophysical and biomechanical properties. MATERIALS AND METHODS
Plant tissue
Both the WT and transgenic alfalfa lines were generated from tissue culture with subsequent production of the transgenic by Reddy et al. (2005)
. The lines were maintained and vegetatively propagated in Washington State University greenhouses. Growing conditions included a light intensity of 150 mmol·m–2·s–1 with a 15 h light/9 h dark cycle at 21° and 16°C, respectively, and a humidity range of 20–35%. Young (4 yr of age) black cottonwood trees [Populus trichocarpa (Torr. et A. Gray)] were grown outdoors on the Washington State University campus, and branches 1 cm in diameter were collected for microscopy.
Lignin analyses
Acetyl bromide and thioacidolysis analyses were carried out as described in Patten et al. (2005)
using three or more vegetatively propagated plants per line.
Dynamic mechanical analyses (modulus of elasticity)
For each WT and pC3H-I alfalfa line, one branch from three plants was collected (N = 3) following 3 wk of growth after cut-back. Internodes 2, 5, and 8 (numbering started from the apical end of the branch) were individually sampled. Internode segments were 25 mm long and were tested in the tension mode using a TRITEC 2000 Dynamic Mechanical Analyzer (Triton Technology, Nottinghamshire, UK). The specimen free length was 10 mm, and the diameter ranged from 1.0 to 1.7 mm. The test consisted of a dynamic strain sweep in the linear viscoelastic range from 0.003% to 0.03% performed at a frequency of 0.5 Hz and at a constant temperature of 30 ± 1°C. To evaluate differences in dynamic moduli, the storage (E') and loss (E'') modulus of each sample was recorded at a 0.02% strain level, and the data were compared with a paired t test using SigmaPlot (San Jose, California, USA) software.
Growth measurements
All plants within each alfalfa line were vegetatively derived from self-stock. For each line, two plants with 8 wk of growth after cut-back were sampled. Branches (not individual plants) were chosen as the experimental unit because branches represent the greatest variability within these lines. Branches derived from crown buds (N = 10) were sampled from each of the WT and pC3H-I lines. Because alfalfa branches grow rapidly and pC3H-I lags in flower development by at least 5 wk relative to WT (see also Reddy et al., 2005
), branches at 8 wk were selected based on the presence of flower buds/flowers to avoid young or axillary branches less than 8 wk of age. The number and lengths of internodes and total stem length were recorded, and their means were analyzed by t tests (
= 0.05) using Microsoft (Richmond, Washington, USA) Excel software.
Lignin histochemistry
Sections were hand-cut from internodes 2, 3, and 8 (IN2, IN3, IN8) of 3-wk-old branches of both WT and pC3H-I lines immediately after the internodes were measured. Sections were directly mounted in phloroglucinol-HCl (Wiesner, 1878
) using standard methods (Ruzin, 1999
) to detect all lignins. For specific S-lignin localization, sections were treated with the Mäule reagent (Mäule, 1901
) as described previously (Patten et al., 2005
).
Gelatinous fiber histochemistry
Gelatinous fibers (gf) were visualized with zinc chloro-iodide (Herzberg reagent) (Scurfield, 1972
); which was formulated using chemicals purchased from Sigma-Aldrich and applied according to the method of Grzeskowiak et al. (1996)
. For imaging at lower magnifications, fresh hand-cut sections from IN2 to the most basal internode of 8-wk-old plants were examined within 20 min of appyling the reagent directly to the section. Gelatinous layers within fibers were identified by a red-purple coloration, in contrast to the orange-brown coloration of surrounding and presumably lignified fiber cell walls (after Scurfield, 1972
).
Additionally, zinc chloro-iodide was used to compare a single mature internode (IN20) from both lines to a branch of black cottonwood. Safranin O and astra blue staining (Srebotnik and Messner, 1994
; Vazquez-Cooz and Meyer, 2002
) were also used to confirm the zinc chloro-iodide visualization results, and phloroglucinol-HCl was used to assess lignification as described earlier. The comparative study used serial cryosections (10 µm thick) obtained from fresh-frozen tissues cut on a Reichert-Jung Cryocut 1800 cryomicrotome (Leica Microsystems, Deerfield, Illinois, USA). Light micrographs were recorded using an Olympus BH-2 light microscope equipped with a ProgRes C12plus digital camera (JENOPTIK, Jena, Germany).
Gelatinous fiber occurrence and distribution
Hand-cut internodal sections were prepared from IN2 to the most basal internode of each branch (N = 10) for each alfalfa line as described for growth measurements. These were then scored for the presence of gelatinous fibers (gf) and nongelatinous, thick-walled fibers (tf), respectively, in primary and secondary reaction tissues using zinc chloro-iodide for visualization (Scurfield, 1972
), as described for gelatinous fiber histochemistry. The sections (475) were imaged in detail at several magnifications using light microscopy as described. After scoring for the presence of xylary gf or tf cells, the means were statistically analyzed with Student's t test. Images of sections from IN5 (i.e., the earliest internode found to have gf cells in either line) to the most basal internode of each branch (10 branches per line) were individually measured for total xylem and total gf cell areas using ImageJ software (Rasband, 1997
) and means were compared with a t test. The extent of tf cell formation was not examined because it was not possible to resolve tf from normal fiber cells at the low magnification required to accurately measure area; zinc chloro-iodide and safranin O/astra blue staining do not differentiate between tf and normal fiber cells.
Cell wall ultrastructure
Identification of reaction vs. normal cell types was further confirmed using transmission electron microscopy (TEM). Samples were harvested from a mature internode (IN20) from both WT and pC3H-I lines, as well as from branch tissue of black cottonwood as a control. Tissues (
5 mm2) were fixed in 2% paraformaldehyde and 1.25% glutaraldehyde in 50 mm piperazine-1,4-bis(2-ethanesulphonic acid) (PIPES) buffer (pH 7.2) overnight at 4°C. Samples were dehydrated using a standard ethanol series, gradually infiltrated with LR White resin (London Resin, Reading, UK), and heat cured. Thin sections were obtained using a diamond knife mounted to a Reichert Ultracut R ultramicrotome (Reichert-Jung GmbH, Heidelberg, Germany), and sections were mounted on formvar-coated 200-mesh nickel grids. Sections were stained with a 3:1 dilution of 4% (w/v) uranyl acetate and 1% (w/v) KMnO4 and observed at 100 kV using a JEOL JEM-1200 EX transmission electron microscope (JEOL, Tokyo, Japan).
RESULTS
Measurement of branch growth and internode elongation
Branches (N = 10) were measured following 8 wk of growth after cut-back. No significant difference was found (P > 0.05) between the total stem lengths and total numbers of internodes for the two alfalfa lines (Table 1). However, the mean internode length was significantly shorter (P < 0.05) for pC3H-I than for WT (Table 1). To examine this further, the single longest internode in each stem was used as a "standard" for growth potential; the longest internode was significantly shorter (P < 0.05) for pC3H-I than WT (Table 1).
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). Histochemical analyses used hand-cut sections treated with either Wiesner or Mäule reagent to localize patterns of presumed lignin deposition (Patten et al., 2005
). Historically, the Wiesner reagent (phloroglucinol-HCl) is used as a general stain (pink to red) to detect p-hydroxycinnamyl aldehyde end-groups in macromolecular lignin of plant tissues. Although frequently cited as specific for coniferyl aldehyde (e.g., Sarkanen and Ludwig, 1971
), the reagent reacts with all three p-hydroxycinnamyl aldehydes (Pomar et al., 2002
; M. Jourdes et al., unpublished manuscript). By contrast, the Mäule reagent is considered specific and is used to differentiate apparent S moieties (red) from the H and G components (brown) (Iiyama and Pant, 1988
; Nakano and Meshitsuka, 1992
; Patten et al., 2005
). Because both reagents give only temporary visualization of color, images were recorded within 20 min of reagent application.
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8.7% lignin, largely of G character, with smaller amounts of H and S moieties (H:G:S ratio of 10:107:22, Table 2). Slightly later in development of the WT vascular tissue (IN3), the cambium (ca) has formed a contiguous circumferential band of cells (Fig. 2B), and lignification has apparently extended throughout the primary medullary or interfascicular ray tissue (pr) and is initiated in the phloem fibers (pf), as demonstrated by the red color with phloroglucinol-HCl (Fig. 2B). This phase of lignification now also apparently involves some detectable S lignin deposition, in at least the pr (light to dark red with the Mäule reagent, Fig. 2H). Again, this is supported by the chemical analyses, which indicated that the overall lignin content had essentially doubled (to 17%) and that the S content increased about five-fold overall, with an H:G:S ratio of 10:110:42.
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26.1%) were close to maturation levels, and the H:G:S ratio 10:229:104 was mainly indicative of differential increases in G and S deposition (Table 2).
pC3H-I alfalfa lignin analyses
Lignification of px in IN2 of pC3H-I was again mainly of the H- or H-/G-type character as visualized by phloroglucinol-HCl (pink-red, Fig. 2D); this lignification was not of the S-type because the Mäule reaction staining was brown rather than red (Fig. 2J). This interpretation was also supported by chemical analyses, which indicated a slightly lower AcBr lignin content in pC3H-I (Table 2). Moreover, the presumably H-lignin-derived, releasable monomeric moieties were only slightly increased relative to WT, whereas the G/S amounts had decreased by at least an order of magnitude (H:G:S ratio of 10:6:0.5). Taken together, these observations suggest that the initial phase of H-lignin deposition is unaffected by pC3H down-regulation as anticipated (Anterola and Lewis, 2002
). Deposition of S-lignin within the pr of IN3 occurs as in WT (Mäule reagent, light red, Fig. 2K and H, respectively) and corresponds well to the chemical analyses, with lignin contents having increased slightly (7.1%) with an H:G:S ratio now of 10:4.3:1.2 (Table 2).
At IN8, the transgenic line (Fig. 2F) was similar to WT (Fig. 2C) in having an apparently uniform deposition of H- and/or H/G-lignins in the pr, sxr, and pf, as well as a faint staining again in the pi by Wiesner reagent. However, the Mäule reagent only gave intense S-lignin staining in the pr cell walls, as evidenced by the distinctive red band adjacent to the pi (Fig. 2L), which had been initiated previously (Fig. 2K). By comparison, a lighter and more uneven coloration of the sxr occurred, indicating a very limited S lignin deposition. The pi also appeared to have a similar pattern of faint staining relative to that of WT. Chemical analyses, in turn, indicated that the lignin contents were now 9.4%, thereby reflecting a very large decrease in overall lignin amount when compared to WT levels (26.1%) (Table 2). Likewise, thioacidolysis analyses again established that while the H-monomeric component dominated, there were small increases in both G and S contents as well (H:G:S ratio of 10:3.5:1.8). Taken together, these data, demonstrate that pC3H-I is, nevertheless, forming small amounts of a primarily H-type lignin during growth and development.
Estimation of alfalfa branch tensile dynamic moduli
The tensile dynamic moduli, storage and loss moduli, were determined for both lines. The storage and loss moduli represent the elastic and viscous components, respectively, of the material properties and provide a measure of viscoelastic behavior. Three-week old branches were sampled at: IN2, with early primary xylem development but little to no fiber development; IN5, where internode elongation is thought to end and secondary growth begins (Vallet et al., 1998
); and IN8, where there is a large volume of lignified secondary xylem growth.
Dynamic moduli were thus determined in triplicate for each internode of both lines and with reasonably good reproducibility (see Fig. 3 for example using pC3H-I). That is, there was a marked increase in storage (E') and loss (E'') moduli with internode development/maturation for both lines, indicating enhanced material properties (Fig. 4). Student's t test, however, showed no significant difference (P = 0.267, 0.116, and 0.810 for internodes 2, 5 and 8, respectively) in the storage modulus (E') between lines WT and pC3H-I. Similarly, the loss modulus (E'') was not significantly different between lines for internodes 2 and 8 (P = 0.979 and 0.225) but was significantly different for internode 5 (P = 0.006) (Fig. 4). At internode 5, the loss modulus apparently is higher for pC3H-I than for WT, but whether this difference is an artifact resulting from the small number of replicates or is a significant difference was not investigated further. In any case, the overall data suggested that despite a range in lignin reduction (i.e., 37% to 64% in IN2 and IN8, respectively, relative to WT levels), the pC3H-I line was capable of maintaining storage and loss modulus properties comparable to those of WT. An explanation for these observations was needed.
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) and was used as the primary histochemical stain in this study. This dye can yield variable results because it does not react directly with wall-bound molecules such as aldehydes in the manner of phloroglucinol-HCL or the Mäule reagent. Instead it intercalates into the cell wall, which can vary in structure and therefore differentially exclude the dye (Grzeskowiak et al., 1996
). This is especially true of transitional cells, which may have variable numbers of secondary cell walls and which also have quite variable histochemical properties when stained with zinc chloro-iodide (Scurfield, 1972
; Grzeskowiak et al., 1996
). Scurfield (1972)
described the G-layer of Eucalyptus subsp. as being "purple-red" (with surrounding walls of yellow), while the general color of transitional cells appeared "yellowish-red," albeit with variability in coloration corresponding to variable cell structure. Grzeskowiak et al. (1996)
also observed a red-purple color in the G-layers of Populus, with the normal cells being yellow. We used these observations to help us interpret staining of alfalfa reaction tissues. Using hand-cut internodal sections made from 10 branches of both lines, we made three interesting observations following zinc chloro-iodide staining (Figs. 5 and 6). First, many of the sections stained positively for gelatinous fibers (gf) as noted in Fig. 5C, E, and G (for IN5 and 7); this staining was even more pronounced in older internodes (see IN20 in Fig. 6A, D). We interpreted this tissue to be reaction tissue in the proper sense. Second, the branch tissues could be visually separated into xylary tissues having either red-purple gf or tissues lacking gf and staining yellow to orange. Third, both plant lines formed thick-walled fibers (tf), which were physically similar to gf cells but stained entirely orange to yellow and were restricted to secondary growth (Figs. 5E–H and 6J, M). The latter two tissue types were interpreted as normal to transitional tissues.
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), the detection of presumed reaction phloem fibers (rpf) cells (Scurfield and Wardrop, 1962
; Wardrop, 1964
; Côté et al., 1969
) allowed us to easily identify reaction tissue in even the youngest internodes lacking gf cells (for example, IN5 in WT, Fig. 5A vs. B, for rpf vs. normal pf, respectively). Additionally, xylem region gf cells were found to occur significantly (P < 0.05) earlier in the primary tissue of the pC3H-I line than WT, i.e., the reaction tissue had developed by IN5 (mean ± SE: 4.70 ± 0.47) in the primary ray tissue of pC3H-I, whereas it occurred later at IN7 (6.70 ± 0.62) in WT (for examples, see Fig. 5E vs. 5C). Results for the secondary reaction tissue were also statistically significant (P < 0.05), with gf cells appearing by IN5 (5.5 ± 0.40) in pC3H-I and by IN7 (7.5 ± 0.65) in WT.
Primary and secondary transitional and normal tissues
Fiber cells (f) of the normal primary tissues of both lines appeared yellow to orange and lacked any detectable G-layers "proper" (i.e., red to purple staining; Fig. 5B, D, F, H). It should be noted that while gf cells were absent, the primary ray tissue in the early internodes (i.e., IN3–5) of both lines still stained a uniform red to orange (for example, see pr of IN5 WT, Fig. 5A, B). This primary ray coloration is likely due to the low lignin contents of the early internodes and was scored as a negative, unless obvious gf cells were present.
Presumed transitional tissues frequently occurred in which gf cells had a typical dark inner layer and light-yellow outer layers (gf in sr, Fig. 5G). Transitional tissues are known to be highly variable (Scurfield and Wardrop, 1963
) and may include tf cells. Moreover, thick-walled fibers (tf) were considered here to constitute transitional tissues in alfalfa, due to their swollen inner walls (similar to gf cells), but they stained orange to yellow (as in normal tissues). Additionally, tf cells first form near gf cells ‘proper' and then extend further around the stem with internode maturity. Interestingly, scoring of tf cells showed the greatest differential (P < 0.05) between the lines, with tf cells appearing by IN5 (5.90 ± 0.35) in the pC3H-I line (for example, Fig. 5E) and later at IN12 (12.50 ± 1.81) for WT (data not shown).
Comparisons of reaction vs. lignin histochemistry
Serial sections were obtained from mature reaction and normal tissues (IN20) of both alfalfa lines and from reaction and normal branch tissue of black cottonwood. The latter was used as a positive control, because cottonwoods are well known to form gf cells (Kaeiser and Boyce, 1965
; Côté et al., 1969
; Isebrands and Bensend, 1972
).
G-layer
Upon staining with zinc chloro-iodide, the G-layers within gf cells in cottonwood were readily observed as red-brown amid a background of orange cell walls (Fig. 6G); IN20 of the WT (Fig. 6A) and pC3H-I (Fig. 6D) lines stained similarly. These results were confirmed by the use of safranin O and astra blue staining. With these stains, the G-layers appeared blue against a pink-red background in all samples examined (Fig. 6B, E, H). Lignin was next localized in the serial sections by use of phloroglucinol-HCl as before for comparative purposes and was detected as a pale pink color because thin (10 µm) serial sections were used. In all samples, and as expected, there was little to no pink coloration in the G-layer, indicating little to no lignin (Fig. 6C, F, I). By contrast, the surrounding cell walls were faint pink, indicating the presence of lignified elements.
Transitional and normal tissues
A few gf cells in cottonwood (Fig. 6G) and the WT line (inset, Fig. 6A) displayed a thin orange coloration internal to the G-layer, almost like an inner membrane (Gim). Similar cells (Gim) were also observed in older internodes (>IN20) from the pC3H-I line (data not shown) and probably represent part of the transitional tissue.
On the other hand, the histochemistry of the thick-walled fibers (tf) in both alfalfa lines appears to be closer to normal cells than to that of the Gim. These were therefore compared in serial section to normal fibers (f) of cottonwood. In the alfalfa tf and the cottonwood f, G-layers of reaction wood "proper" were considered to be absent if zinc chloro-iodide staining did not produce a red to purple-brown color (Fig. 6J, M, P), or if safranin O and astra blue double staining did not produce characteristic blue cell wall layers (Fig. 6K, N, Q). There was, however, a small amount of blue coloration around the lumens of the tf in the pC3H-I line (Fig. 6N), which may reflect either decreased lignification or a minor artifact of double staining. In any case, no G-layers (as in reaction wood ‘proper') were observed in the tf of this line or in the other samples. Finally, phloroglucinol-HCl histochemistry was similar among all plant samples with tf, wherein the pink coloration, indicative of lignification, was present throughout the cell walls (Fig. 6L, O, R).
Quantification of xylem and xylem reaction tissue areas
Images of the tissue sections were digitally measured using ImageJ software as described in the Materials and Methods. Images of internodes 2 to 4 were excluded because they generally lack gf cells. The pC3H-I line formed significantly (P < 0.05) more reaction tissue and total xylem tissue than WT (Table 3).
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) was also clearly visible in both alfalfa lines and in cottonwood (black arrowheads, Fig. 7A, D, F).
It should be noted, however, that the thickness of the G-layer was variable even among adjacent fiber cells within each plant line and species (data not shown), in agreement with previous reports (Côté et al., 1969
; Isebrands and Bensend, 1972
). Furthermore, all three samples displayed the characteristic artifacts of G-layers, involving a slight detachment from the S2 subwall layer (double arrowheads, Fig. 7A) and tissue folding upon interaction with grid coating (seen as a black line in the G-layer, Fig. 7A, D, F); such preparation artifacts have been reported many times (Côté et al., 1969
; Clair et al., 2005
).
"Normal" fibers
By contrast, the normal fibers (f) of all tissues examined had the expected three secondary subwall layers (S1, S2, and S3) (Fig. 7B, G), which also varied in thickness. A thick-walled fiber (tf) cell is also clearly visible in the WT (Fig. 7E).
Vessels
Finally, vessel elements were imaged from the same sections and grids to confirm that secondary tissue was examined and to compare the cell wall structure of vessel elements and fiber cells. The vessels (v) in all samples displayed the expected secondary cell wall architecture, including three lignified subwall layers, S1, S2, S3 (v, Fig. 7C, E, H). Additionally, the vessels stained more intensely than the fiber cell walls, especially in S2, presumably because of differential lignin content or compositions of these cell wall types (for example, see Fig. 7H).
Other general characteristics of reaction tissue in alfalfa
Besides the gf "proper" (which may or may not be present in some species), other characteristics of reaction wood were considered.
Spiral patterning of reaction tissue
Older branch internodes (>IN12) of both alfalfa lines also contained reaction tissue in spiral or banded patterns (data not shown). These patterns nearly circumscribed the woody crown tissue of both lines. Such patterns are likely related to the rapid growth and the subsequent tensile stress gradient thus generated around the stems in both lines during growth and development, with more stresses experienced or perceived by the pC3H down-regulated line because of its reduced lignin content. Spiral reaction wood patterns have also been observed for saplings and rapidly growing trees (Barnett and Jeronimidis, 2003
).
Variable eccentric growth
Because eccentric growth (i.e., growth greater on one side than the other) frequently occurs on the upper side (tension wood) of angiosperm tree branches (Wardrop, 1964
), it was instructive to determine whether similar histology occurred in alfalfa branches. While some branch samples displayed a larger radius where gf cells occurred, others either lacked eccentric growth but had reaction tissue or had elongated eccentric growth that only somewhat correlated to spiral patterned reaction tissue (data not shown). Given the variability noted, however, eccentric growth was considered an unreliable characteristic of reaction tissue in alfalfa. This is consistent with previous observations that shrub species do not usually form eccentric growth in relation to reaction tissue (Wardrop, 1964
).
DISCUSSION
The pC3H gene is involved in regulation of carbon flux into the G and S segments during macromolecular lignin assembly (Anterola and Lewis, 2002
; Anterola et al., 2002
). Reduction of pC3H activity would thus be expected to result in a lignin-reduced and p-hydroxycinnamyl alcohol (H)-enriched phenotype. This is because the H-phase of lignification precedes deposition of both G and S moieties (Fergus and Goring, 1970a
, b
; Whiting and Goring, 1982
; Terashima and Fukushima, 1988
; Fukushima and Terashima, 1991
). Indeed, as expected, downregulating or mutating pC3H in A. thaliana results in a depleted lignin phenotype (64% less lignin than WT at maturation; M. Jourdes et al., unpublished manuscript) that contains mostly H-units. However, this plant line was also severely dwarfed because of unknown "pleiotropic" effects, and its vascular apparatus was greatly impaired (M. Jourdes et al., unpublished manuscript; see also Franke et al., 2002
).
In the case of alfalfa, 11 lines downregulated in pC3H activity were generated, and line pC3H-I was selected for further study because it had overall the lowest lignin content (9.4%), i.e., corresponding to a circa 64% reduction in lignin. Moreover, pC3H-I predictably had greatly reduced S/G levels in its lignin, such that the lignin was mainly of the H-character. It is likely that the effects of this genetic manipulation are continually perceived and responded to during pC3H-I growth and development, i.e., ultimately resulting in global gene expression changes. That is, lacking an ability to preferentially form vascular tissues with normal lignin content and composition, the pC3H-I stem vascular tissue developed differently. In fact the pC3H-I line displayed a 5 wk delay in flowering (Fig. 8).
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), 10 branches per line were sampled to allow for statistical comparisons. Because the average internode length was shorter for pC3H-I and internode elongation is a feature of primary growth, it follows that pC3H-I begins secondary growth earlier than WT. This may in part account for why pC3H-I forms gf and tf cells earlier than does the WT and also forms more xylem tissue and xylem reaction tissues (with gf cells). In fact, early cessation of internode elongation is well documented in numerous angiosperm species in response to mechanical challenges, such as controlled bending, shaking, or brushing (Mitchell, 1996
; Garner and Björkman, 1997
, 1999
; Coutand and Moulia, 2000
; Coutand et al., 2000
). Perhaps this apparent reduction in internodal elongation is also induced by perception of potential weakening of the overall vascular apparatus through the reduction in lignin content.
Differential patterns of lignin deposition
One noteworthy difference in histochemical staining for lignin was found between the alfalfa lines and suggested that reduction of lignin content in pC3H-I was more specific to secondary growth (Fig. 2).
While the reasons for the apparent qualitative histochemical differences in S-lignin deposition between the pr/pf and the sxr region of pC3H-I remain to be established, one possibility is that deposition is differentially regulated between primary and secondary growth in alfalfa. It is well-known that the deposition of specific lignin types is regulated both spatially and temporally. For instance in angiosperms, H deposition precedes G in xylem and vessels, followed by H to G to S in vessels and fibers. On the other hand, we have not ruled out the possibility that observed differences in patterns of lignin deposition may be due to specific promoter effects introduced by use of the heterologous pal2 bean promoter. These observations thus underscore the complexity of studying lignification proper in planta.
Material properties of alfalfa
In terms of physiological function, reaction woody tissue formation provides a mechanism to respond to changes in perceived longitudinal stress (see Pilate et al., 2004
), i.e., via formation of lignin-deficient G-layers within the gf (Norberg and Meier, 1966
; Côté et al., 1969
). These include structural stresses, such as axial displacement (bending) (Wardrop, 1964
; Scurfield, 1973
; Barnett and Jeronimidis, 2003
), internal axial stresses (i.e., rapidly growing upright stems) (Isebrands and Bensend, 1972
; Fisher and Stevenson, 1981
; Barnett and Jeronimidis, 2003
), as well as those introduced by the twining habit of some vines (Meloche and Vaughn, 2006
). Given the mechanical support provided by this tissue type, angiosperm reaction tissues are also known to be cellulose rich and lignin deficient.
Lignin deficiency in numerous genetically modified plant lines causes weakness in stems and vascular integrity as compared to the corresponding WT lines (reviewed by Anterola and Lewis, 2002
). For instance, a transgenic tobacco line, downregulated in cinnamyl alcohol dehydrogenase (CAD) activity, had a lower longitudinal tensile modulus than did the WT (Hepworth and Vincent, 1998
). Additionally, a double mutation knockout of CAD genes in A. thaliana resulted in an extensively depleted lignin phenotype, with a tendency for the stems to become prostrate (Sibout et al., 2005
; Jourdes et al., 2007
). The stem sections, however, were also significantly reduced in storage tensile modulus (Jourdes et al., 2007
), in keeping with the observed phenotype.
Thus, the comparable tensile dynamic moduli observed for both WT and the pC3H-I downregulated line suggests that a compensatory mechanism may help maintain structural properties. Specifically, the increased formation of gf and tf cells in the pC3H-I line may in part compensate for the decreased lignin contents and thus prevent decreases in structural support and vascular integrity which would otherwise result. However, the validity of this proposed mechanism will require detailed analyses of various lines containing different amounts of lignin (i.e., intermediary between WT and the pC3H-I line).
Reaction tissue
Woody plants have a mechanism for controlling both branch orientation and restoration of vertical alignment when stems are displaced from the normal vertical alignment. In both gymnosperms and angiosperms, this mechanism leads to formation of various tissues, collectively referred to as reaction wood, which is frequently separated from normal wood by so-called transitional wood with intermediate characteristics. In gymnosperms, reaction tissue is termed compression wood and contains increased H-lignin content (Timell, 1986; Fukushima and Terashima, 1991
). While reaction wood has been considered to result from differences in gravity perception (i.e., via signaling and transduction), this process can be duplicated in microgravity through mechanical bending (Kwon et al., 2001
). It is poorly understood though how these stress signals are perceived and how the signal cascade leading to reaction tissue formation is transduced.
Woody angiosperms have a distinct, but somewhat complementary, mechanism leading to another form of reaction tissue known as tension wood. In general, tension wood is characterized by much lower lignin contents, as well as reduced number and diameter of vessel elements (Wardrop, 1964
; Kucera and Philipson, 1977
). However, other characteristics of woody angiosperm reaction tissue are highly variable, including whether gelatinous fibers (gf) and/or eccentric growth patterns (due to differing radii between upper and lower sides of the branch) occur (Wardrop, 1964
; Kucera and Philipson, 1977
). Nevertheless, the gf cell type has frequently been used as a diagnostic character of tension wood and is distinguished by a gelatinous (G-) layer, which has a gel-like appearance under magnification and often replaces one or more subwall layers of the fiber secondary cell wall (Wardrop, 1964
; Carlquist, 2001
). Furthermore, the higher tensile strength and Young's modulus found in tension wood relative to normal wood (Barnett and Jeronimidis, 2003
; Clair et al., 2003
) is thought to be partly due to the high tensile strength generated by orientation of the cellulose microfibrils within the G-layer (Yamamoto et al., 1993
; Prodhan et al., 1995
). It should also be noted that at least three "fiber" cell types (tracheids, fiber tracheids, and libriform fibers) occur in dicotyledons and can form G layers under tensile stress (Carlquist, 2001
).
Reaction tissue identification and differential formation in alfalfa
Zinc chloro-iodide stained gelatinous fibers "proper" red-purple in both alfalfa lines and cottonwood reaction tissue consistent with previous studies of reaction wood in trees (Scurfield, 1972
; Grzeskowiak et al., 1996
). Results from safranin O/astra blue histochemistry and TEM confirmed the presence of gf cells.
With positive identification of reaction tissue in alfalfa (WT and the pC3H-I lines), we sought to determine whether reaction tissue formation was more extensive in pC3H-I than in WT, possibly allowing pC3H-I to compensate for lost mechanical strength following lignin downregulation. Results showed that initiation of gf formation in pC3H-I begins on average two internodes earlier than WT and is possibly related to an apparently earlier cessation of internode elongation (earlier secondary growth) in the pC3H-I. Additionally, the pC3H-I was shown to form more total xylem area and more total reaction xylem area than the WT. These data may help in part explain the phenomenological observation of Reddy et al. (2005)
that pC3H downregulated alfalfa lines appeared to produce higher amounts of cellulose. It is still not clear whether increased reaction tissue formation in pC3H-I is a sole and direct effect of reduced lignin contents or whether there are additional effects resulting from genetic manipulation. Future work will address this issue using other transgenic pC3H downregulated alfalfa lines as indicated earlier.
Transitional tissue identification and differential formation in alfalfa
Besides gf cells "proper," small groups of similar cells with an "inner membrane" were observed in both alfalfa lines and cottonwood. Fibers with a similar inner membrane have been previously reported and proposed to be lignified (Scurfield and Wardrop, 1963
; Scurfield, 1972
; Joseleau et al., 2004
; Gierlinger and Schwanninger, 2006
). However, we were not able to confirm lignification of this inner layer by phloroglucinol-HCl staining. Furthermore, while Gim was generally uncommon in both alfalfa lines and the cottonwood sample, the number of Gim cells increased as alfalfa tissues became more woody (data not shown). Such cell types may be related to transitional tissue (i.e., between reaction and normal tissue (Scurfield and Wardrop, 1963
).
Additionally, we observed thick-walled fibers (tf) directly in contact with gf cells "proper," as well as developing later throughout the areas of stems lacking gf cells "proper." The tf cells appeared similar to the swollen, irregularly shaped gf cells but staining was variable (orange to yellow by zinc chloro-iodide), perhaps indicating a cell wall chemistry intermediate to that of reaction tissue (which stains predominately red-purple) and normal tissue (which stains yellow). These cells seem to correspond with Scurfield's (1972) reports of transitional tissue staining variably with zinc chloro-iodide. He reasoned that the histochemical variability was probably due to the variable ultrastructure of such transitional fibers, which had variable numbers of secondary cell walls in addition to a G-layer of variable width. In alfalfa preliminary descriptions of xylary tf cells have been reported by Vallet et al. (1996)
and Engels and Jung (1998)
but without comment on their physiological roles or prevalence. It is very likely that the tf cells constitute transitional tissues, but it is also recognized that the inner cell wall swelling of both gf and tf cells may be exaggerated because of artifact generated during sample preparation (Côté et al., 1969
; Clair et al., 2005
).
Formation of tf cells occurred seven internodes later in WT than in the pC3H-I. Although it was not possible to accurately calculate the area of tf cells, the observation that this cell type occurs so much earlier in pC3H-I indicates that tf cells (transitional tissues) are induced in the pC3H-I at the same time as gf cells. Presumably, WT possesses enough mechanical strength to support all but the most basal internodes of the branch without tf cell formation in addition to gf cells.
Concluding remarks
To our knowledge, this represents the first description of reaction tissue in alfalfa. The alfalfa growth habit itself suggests a capacity for its formation, i.e., because woody alfalfa tissue is formed in the basal portion of the plant (the "crown") in order to survive winter (Undersander et al., 1997
). Additionally, alfalfa branches elongate rapidly with variable displacement from a vertical alignment. Reaction tissue has in fact, been reported in numerous woody members of the family Fabaceae (to which alfalfa belongs) (Onaka, 1949
; Ne
esan
, 1955
; Wardrop, 1964
; Höster and Liese, 1966
; Fisher and Stevenson, 1981
).
It can thus be provisionally anticipated that many, if not all, lignin-reduced woody gymnosperms and angiosperms, will form reaction or transitional wood, i.e., the organism will use other normal mechanisms to reinforce branches and leaning stems. In possible agreement with this, several transgenic poplar trees downregulated for CAD activity (downstream of pC3H) were also suspected to have an altered ability to form G-layers, although no data were reported (Pilate et al., 2004
). If correct, as predicted herein, this process would be undesirable for many forestry/pulp and paper applications because the gelatinous fibers have poor strength quality due to decreased bonding properties (Dickison, 2000
).
Nevertheless, how these biochemical processes are initiated and result in the formation of reaction tissue (how does the plant perceive the need for its formation and then activate the various biochemical pathways involved?), are very important questions that remain to be fully delineated. Indeed, obtaining a detailed biochemical understanding of the factors controlling reaction wood formation in fast growing trees and other species is obviously important to agriculture and the forestry industry.
FOOTNOTES
1 The authors extend thanks to C. Davitt and V. Lynch-Holm of the Electron Microscopy and Imaging Center, Washington State University (WSU) for technical advice on TEM; and to M. F. Wiater, Department of VCAPP, Washington State University, on statistical analyses related to alfalfa growth and gelatinous fiber measurements. Appreciation is also extended to C. Whitney, IBC, Washington State University, for maintenance and growth of the pC3H downregulated and WT lines. Lines were prepared by the Noble Foundation using standard transgenic approaches as a subcontract to N.G.L. with funding from the DOE. Thanks also for the generous support by the U.S. Department of Energy (DE-FG-0397ER20259), National Science Foundation (MCB-0417291), McIntire Stennis, and the G. Thomas and Anita Hargrove Center for Plant Genomic Research. 
4 Author for correspondence (e-mail: lewisn{at}wsu.edu
)
LITERATURE CITED
Anterola A. M. Jeon J.-H. Davin L. B. Lewis N. G.. 2002. Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism. Journal of Biological Chemistry 277: 18272-18280..
Anterola A. M. Lewis N. G.. 2002. Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry 61: 221-294..[CrossRef][Web of Science][Medline]
Barnett J. R. Jeronimidis G.. 2003. Reaction wood. In J. R. Barnett, G. Jeronimidis [eds.] Wood quality and its biological basis, 118-136. Blackwell, Oxford, UK..
Carlquist S.. 2001. Comparative wood anatomy: systematic, ecological, and evolutionary aspects of dicotyledon wood. Springer-Verlag, Berlin, Germany..
Clair B. Gril J. Baba K. Thibaut B. Sugiyama J.. 2005. Precautions for the structural analysis of the gelatinous layer in tension wood. International Association of Wood Anatomists Journal 26: 189-195..
Clair B. Ruelle J. Thibaut B.. 2003. Relationship between growth stress, mechanical–physical properties and proportion of fibre with gelatinous layer in chestnut (Castanea sativa Mill.). Holzforschung 57: 189-195..[CrossRef][Web of Science]
Côté W. A. Jr. Day A. C. Timell T. E.. 1969. A contribution to the ultrastructure of tension wood fibers. Wood Science and Technology 3: 257-271..[CrossRef][Web of Science]
Coutand C. Julien J. L. Moulia B. Mauget J. C. Guitard D.. 2000. Biomechanical study of the effect of a controlled bending on tomato stem elongation: global mechanical analysis. Journal of Experimental Botany 51: 1813-1824..
Coutand C. Moulia B.. 2000. Biomechanical study of the effect of a controlled bending on tomato stem elongation: local strain sensing and spatial integration of the signal. Journal of Experimental Botany 51: 1825-1842..
Davin L. B. Lewis N. G.. 2005. Lignin primary structures and dirigent sites. Current Opinion in Biotechnology 16: 407-415..[CrossRef][Web of Science][Medline]
Dickison W. C.. 2000. Integrative plant anatomy. Harcourt Academic Press, Burlington, Massachussetts, USA..
Engels F. M. Jung H. G.. 1998. Alfalfa stem tissues: cell-wall development and lignification. Annals of Botany 82: 561-568..
Fergus B. J. Goring D. A. I.. 1970a. The distribution of lignin in birch wood as determined by ultraviolet microscopy. Holzforschung 24: 118-124..[Web of Science]
Fergus B. J. Goring D. A. I.. 1970b. The location of guaiacyl and syringyl lignins in birch xylem tissue. Holzforschung 24: 113-117..[Web of Science]
Fisher J. B. Wassmer Stevenson J.. 1981. Occurrence of reaction wood in branches of dicotyledons and its role in tree architecture. Botanical Gazette 142: 82-95..
Franke R. Hemm M. R. Denault J. W. Ruegger M. O. Humphreys J. M. Chapple C.. 2002. Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis. Plant Journal 30: 47-59..[CrossRef][Web of Science][Medline]
Fukushima K. Terashima N.. 1991. Heterogeneity in formation of lignin. Part XV. Formation and structure of lignin in compression wood of Pinus thunbergii studied by microautoradiography. Wood Science and Technology 25: 371-381..[Web of Science]
Garner L. C. Björkman T.. 1997. Using impedance for mechanical conditioning of tomato transplants to control excessive stem elongation. HortScience 32: 227-229..
Garner L. C. Björkman T.. 1999. Mechanical conditioning of tomato seedlings improves transplant quality without deleterious effects on field performance. HortScience 34: 848-851..
Gierlinger N. Schwanninger M.. 2006. Chemical imaging of poplar wood cell walls by confocal Raman microscopy. Plant Physiology 140: 1246-1254..
Grzeskowiak V. Sassus F. Fournier M.. 1996. Coloration macroscopique, retraits longitudinaux de maturation et de séchage du bois de tension du peuplier (Populus x euramericana cv I.214). Annales des Sciences Forestières 53: 1083-1097..[CrossRef][Web of Science]
Heller W. Kühnl T.. 1985. Elicitor induction of a microsomal 5-O-(4 coumaroyl) shikimate 3'-hydroxylase in parsley cell suspension cultures. Archives of Biochemistry and Biophysics 241: 453-460..[CrossRef][Web of Science][Medline]
Hepworth D. G. Vincent J. F. V.. 1998. The mechanical properties of xylem tissue from tobacco plants (Nicotiana tabacum ‘Samsun'). Annals of Botany 81: 751-759..
Höster H.-R. Liese W.. 1966. Über das Vorkommen von Reaktionsgewebe in Wurzeln und Ästen der Dikotyledonen. Holzforschung 20: 80-90..[Web of Science]
Iiyama K. Pant R.. 1988. The mechanism of the Mäule colour reaction. Introduction of methylated syringyl nuclei into softwood lignin. Wood Science and Technology 22: 167-175..[CrossRef][Web of Science]
Isebrands J. G. Bensend D. W.. 1972. Incidence and structure of gelatinous fibers within rapid-growing eastern cottonwood. Wood and Fiber 4: 61-71..
Joseleau J.-P. Imai T. Kuroda K. Ruel K.. 2004. Detection in situ and characterization of lignin in the G-layer of tension wood fibres of Populus deltoides. Planta 219: 338-345..[CrossRef][Web of Science][Medline]
Jourdes M. Cardenas C. L. Laskar D. D. Moinuddin S. G. A. Davin L. B. Lewis N. G.. 2007. Plant cell walls are enfeebled when attempting to preserve native lignin configuration with poly-p-hydroxycinnamaldehydes: evolutionary implications. Phytochemistry: in press..
Kaeiser M. Boyce S. G.. 1965. The relationship of gelatinous fibers to wood structure in eastern cottonwood (Populus deltoides). American Journal of Botany 52: 711-715..[CrossRef][Web of Science]
Kucera L. J. Philipson W. R.. 1977. Occurrence of reaction wood in some primitive dicotyledonous species. New Zealand Journal of Botany 15: 649-654..[Web of Science]
Kwon M. Bedgar D. L. Piastuch W. Davin L. B. Lewis N. G.. 2001. Induced compression wood formation in Douglas fir (Pseudotsuga menziesii) in microgravity. Phytochemistry 57: 847-857..[CrossRef][Web of Science][Medline]
Laskar D. D. Jourdes M. Patten A. M. Helms G. L. Davin L. B. Lewis N. G.. 2006. The Arabidopsis cinnamoyl CoA reductase irx4 mutant has a delayed but coherent (normal) program of lignification. Plant Journal 48: 674-686..[CrossRef][Web of Science][Medline]
Lewis N. G. Davin L. B. Sarkanen S.. 1999. The nature and function of lignins. In Sir D. H. R. Barton, K. Nakanishi, O. Meth-Cohn [eds.] Comprehensive natural products chemistry, vol. 3, 617-745. Carbohydrates and their derivatives including tannins, cellulose and related lignins. Elsevier Science, London, UK..
Mäule C.. 1901. Das Verhalten verholzter Membranen gegen Kaliumpermanganat, eine Holzreaktion neuer Art. Beiträge zur wissenschaftlichen Botanik 4: 166-185..
Meloche C. G. Vaughn K. C.. 2006. Gelatinous fibers are critical in coiling of tendrils and twining of vines. Botany 2006: annual conference of the Botanical Society of America, Chico, California, USA, 2006, Abstract 116. Website http://www.2006.botanyconference.org/engine/search/index.php?func=detail&aid=116..
Mitchell C. A.. 1996. Recent advances in plant responses to mechanical stress: theory and application. HortScience 31: 31-35..[Medline]
Nakano J. Meshitsuka G.. 1992. The detection of lignin. In S. Y. Lin, C. W. Dence [eds.] Methods in lignin chemistry, 23-32. Springer-Verlag, Berlin, Germany..
Neesan V.. 1955. Occurrence of the reaction wood from the taxonomic point of view. Sborník Vysoké 
koly Z
mdelské a Lesnické Fakulty v Brn 3: 131-149..
Norberg P. H. Meier H.. 1966. Physical and chemical properties of the gelatinous layer in tension wood fibers of aspen (Populus tremula L.). Holzforschung 20: 174-178..[Web of Science]
Onaka F.. 1949. Studies on compression and tension wood. Wood Research: Bulletin of the Wood Research Institute, Kyoto University 1: 1-88..
Patten A. M. Cardenas C. L. Cochrane F. C. Laskar D. D. Bedgar D. L. Davin L. B. Lewis N. G.. 2005. Reassessment of effects on lignification and vascular development in the irx4 Arabidopsis mutant. Phytochemistry 66: 2092-2107..[CrossRef][Web of Science][Medline]
Pilate G. Chabbet B. Cathala B. Yoshinaga A. Leplé J.-C. Laurans F. Lapierre C. Ruel K.. 2004. Lignification and tension wood. Comptes Rendus Biologies 327: 889-901..[CrossRef][Web of Science][Medline]
Pomar F. Merino F. Ros Barceló A.. 2002. O-4-Linked coniferyl and sinapyl aldehydes in lignifying cell walls are the main targets of the Wiesner (phloroglucinol-HCl) reaction. Protoplasma 220: 17-28..[CrossRef][Web of Science][Medline]
Prodhan A. K. M. A. Funada R. Ohtani J. Abe H. Fukazawa K.. 1995. Orientation of microfibrils and microtubules in developing tension-wood fibres of Japanese ash (Fraxinus mandshurica var. japonica). Planta 196: 577-585..[Web of Science]
Rasband W. S.. 1997–2006. ImageJ software: version 1.36b. U.S. National Institutes of Health, Bethesda, Maryland, USA. Website http://rsb.info.nih.gov/ij/ [accessed 13 March 2006]..
Reddy M. S. S. Chen F. Shadle G. Jackson L. Aljoe H. Dixon R. A.. 2005. Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa (Medicago sativa L.). Proceedings of the National Academy of Sciences, USA 102: 16573-16578..
Ruzin S. E.. 1999. Plant microtechnique and microscopy. Oxford University Press, New York, New York, USA..
Sarkanen K. V. Ludwig C. H.. 1971. Definition and nomenclature. In K. V. Sarkanen, C. H. Ludwig [eds.] Lignins: occurrence, formation, structure and reactions, 1-18. Wiley, New York, New York, USA..
Schoch G. Goepfert S. Morant M. Hehn A. Meyer D. Ullmann P. Werck-Reichhart D.. 2001. CYP98A3 from Arabidopsis thaliana is a 3'-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. Journal of Biological Chemistry 276: 36566-36574..
Scurfield G.. 1972. Histochemistry of reaction wood cell walls in two species of Eucalyptus and in Tristania conferta R. BR. Australian Journal of Botany 20: 9-26..[CrossRef][Web of Science]
Scurfield G.. 1973. Reaction wood: its structure and function. Lignification may generate the force active in restoring the trunks of leaning trees to the vertical. Science 179: 647-655..
Scurfield G. Wardrop A. B.. 1962. The nature of reaction wood. VI. The reaction anatomy of seedlings of woody perennials. Australian Journal of Botany 10: 93-105..[CrossRef]
Scurfield G. Wardrop A. B.. 1963. The nature of reaction wood. VII. Lignification in reaction wood. Australian Journal of Botany 11: 107-116..[CrossRef]
Sibout R. Eudes A. Mouille G. Pollet B. Lapierre C. Jouanin L. Séguin A.. 2005. Cinnamyl alcohol dehydrogenase-C and -D are the primary genes involved in lignin synthesis in the floral stem of Arabidopsis. Plant Cell 17: 2059-2076..
Srebotnik E. Messner K.. 1994. A simple method that uses differential staining and light microscopy to assess the selectivity of wood delignification by white rot fungi. Applied and Environmental Microbiology 60: 1383-1386..
Terashima N. Fukushima K.. 1988. Heterogeneity in formation of lignin. XI. An autoradiographic study of the heterogeneous formation and structure of pine lignin. Wood Science and Technology 22: 259-270..[CrossRef][Web of Science]
Timmel T. E.. 1986. Compression wood in gymnosperms. Springer-Verlag, New York, New York, USA..
Undersander D. Vassalotti P. Cosgrove D.. 1997. Alfalfa: germination and growth. Publication A3681. University of Wisconsin Cooperative Extension Publishing, Madison, Wisconsin, USA..
Vallet C. Chabbert B. Czaninski Y. Monties B.. 1996. Histochemistry of lignin deposition during sclerenchyma differentiation in alfalfa stems. Annals of Botany 78: 625-632..
Vallet C. Lemaire G. Monties B. Chabbert B.. 1998. Cell wall fractionation of alfalfa stem in relation to internode development: biochemistry aspect. Journal of Agricultural and Food Chemistry 46: 3458-3467..[CrossRef][Web of Science]
Vazquez-Cooz I. Meyer R. W.. 2002. A differential staining method to identify lignified and unlignified tissues. Biotechnic and Histochemistry 77: 277-282..[CrossRef]
Wardrop A. B.. 1964. The reaction anatomy of arborescent angiosperms. In M. H. Zimmermann [ed.] The formation of wood in forest trees, 405-456. Academic Press, New York, New York, USA..
Whiting P. Goring D. A. I.. 1982. Chemical characterization of tissue fractions from the middle lamella and secondary wall of black spruce tracheids. Wood Science and Technology 16: 261-267..[CrossRef][Web of Science]
Wiesner J.. 1878. Note über das Verhalten des Phloroglucins und einiger verwandter Körper zur verholzten Zellmembran. Sitzungsberichte der Kaiserlichen Akademie der Wissenschaften. Math. Nat. Classe 77: 60-66..
Yamamoto H. Okuyama T. Yoshida M.. 1993. Generation process of growth stresses in cell walls. V. Model of tensile stress generation in gelatinous fibers. Mokuzai Gakkaishi 39: 118-125..[Web of Science]
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