Download A xylem-specific cellulose synthase gene from aspen (Populus

The Plant Journal (2000) 22(6), 495±502
A xylem-speci®c cellulose synthase gene from aspen
(Populus tremuloides) is responsive to mechanical stress
Luguang Wu², Chandrashekhar P. Joshi and Vincent L. Chiang*
Plant Biotechnology Research Center, School of Forestry and Wood Products, Michigan Technological University,
Houghton, MI 49931, USA
Received 3 December 1999; revised 25 February 2000; accepted 20 March 2000.
*For correspondence (fax +1 906 487 2915; e-mail [email protected]).
²
Present address: Department of Botany, Queensland University, Brisbane, Queensland 4072, Australia.
Summary
Angiosperm trees accumulate an elevated amount of highly crystalline cellulose with a concomitant
decrease in lignin in the cell walls of tension-stressed tissues. To investigate the molecular basis of this
tree stress response, we cloned a full-length cellulose synthase (PtCesA) cDNA from developing xylem of
aspen (Populus tremuloides). About 90% sequence similarity was found between the predicted PtCesA
and cotton GhCesA proteins. Northern blot and in situ hybridization analyses of PtCesA gene transcripts
in various aspen tissues, and PtCesA gene promoter-b-glucuronidase (GUS) fusion analysis in transgenic
tobacco, demonstrated conclusively that PtCesA expression is con®ned to developing xylem cells during
normal plant growth. During mechanical stress induced by stem bending, GUS expression remained in
xylem and was induced in developing phloem ®bers undergoing tension stress, but was turned off in
tissues undergoing compression on the opposite side of the bend. Our results suggest a unique role for
PtCesA in cellulose biosynthesis in both tension-stressed and normal tissues in aspen, and that the on/
off control of PtCesA expression may be a part of a signaling mechanism triggering a stress-related
compensatory deposition of cellulose and lignin that is crucial to growth and development in trees.
Introduction
In response to mechanical and gravitational stimuli sensed
in non-vertically growing stems, angiosperm trees undergo corrective growth during which tension wood develops
on the upper side of their leaning stems (Timell, 1986).
Tension wood ®bers are known for the deposition in their
lumen of a gelatinous or G-layer, which consists almost
exclusively of the axially oriented cellulose micro®brils
with an exceptionally high crystallinity (Norberg and
Meier, 1966). Xylem and phloem ®ber walls in tension
wood also exhibit increased levels of highly crystalline
cellulose (Cote et al., 1969) and decreased levels of lignin
(Timell, 1986). Thus massive synthesis of highly ordered
cellulose in tension wood has been thought to be part of a
stress-sensing mechanism leading to increased mechanical support in angiosperm trees (Timell, 1986). However,
because of the unavailability of tree cellulose synthase
genes, the underlying molecular mechanisms of this
remarkable cellulose biosynthesis-based stress response
in trees are unknown.
Our current understanding of the molecular mechanism
of cellulose biosynthesis in higher plants is mainly derived
ã 2000 Blackwell Science Ltd
from studies in model herbaceous plants and ®ber crops.
Cellulose, a linear polymer of b-1,4-glucan residues, is
formed from UDP-glucose and this reaction is catalyzed by
the enzyme cellulose synthase (CesA, EC 2.1.4.12) (Delmer
and Amor, 1995; Saxena et al., 1990; for review and new
nomenclature see Delmer, 1999). The ®rst CesA gene was
cloned from bacteria that produce extracelluar cellulose
(Matthysse et al., 1995; Saxena et al., 1990; Wong et al.,
1990). By searching for UDP-glucose binding motifs,
suggested to be conserved in processive glycosyltransferases (Saxena et al., 1995), Pear et al. (1996) isolated the
®rst putative higher plant CesA cDNA (GhCesA, GenBank
accession number GHU58283) from cotton ®bers with
active secondary wall cellulose synthesis. The GhCesA
gene was thought to be associated with the biosynthesis
of highly crystalline cellulose in secondary cell walls of
developing cotton ®bers. Arabidopsis mutants, rsw1
(radial swelling) defective in crystalline cellulose deposition (Baskin et al., 1992), and irx3 (irregular xylem)
defective in overall cellulose production (Turner and
Somerville, 1997), provided a genetic base for isolating
495
496 Luguang Wu et al.
content and position criteria for plant polyadenylation
signals (Joshi, 1987b). Comparison of the nucleotide
sequence of PtCesA with currently known full-length
higher plant CesA cDNAs from Arabidopsis (Arioli et al.,
1998; Taylor et al., 1999; Wu et al., 1998); cotton (Pear et al.,
1996); and hybrid poplar (Wang and Loopstra, 1998),
showed 61±63, 78 and 64% identity, respectively. Amino
acid sequence comparison indicated that PtCesA exhibits a
60±65% identity and 67±73% similarity to the CesA
polypeptides encoded by the Arabidopsis and hybrid
poplar CesA cDNAs. PtCesA shows the highest identity/
similarity (86/90) to cotton GhCesA, believed to be a
secondary cell wall-speci®c CesA (Pear et al., 1996).
Recently, Delmer (1999) suggested that plant CesA
proteins have a highly conserved spatial arrangement of
certain sequence regions, constituting a higher plantspeci®c conserved domain structure. Four plant-speci®c
conserved regions (CR-Ps, Figure 1) are found to be
interspersed between three conserved H regions (H-1 to
H-3). All known plant CesAs contain two hypervariable
regions (HVR-1 and HVR-2) with no signi®cant sequence
conservation. However, the arrangement of these two
hypervariable regions in plant CesA proteins is conserved
± one near the N-terminal and the other between the H-2
and H-3 regions. PtCesA has ®ve CR-P, three H, and two
HVR regions, and their spatial arrangement is consistent
with the plant-speci®c conserved domain structure (Figure
1). The three conserved D residues and QXXRW motif
present in many processive glycosyltransferases (Saxena
et al., 1995) are also present in the central, putatively
cytoplasmic domain of the PtCesA protein.
Hydropathy plot analysis indicates that the PtCesA
protein has eight transmembrane binding domains ± two
near the amino terminal and six at the carboxyl terminal
(Figure 1). This is consistent with the predicted molecular
con®guration shared by known CesA proteins (Arioli et al.,
1998; Pear et al., 1996; Taylor et al., 1999). The 200 amino-
additional plant CesA genes. Arioli et al. (1998) and Taylor
et al. (1999) then mapped and cloned the Arabidopsis CesA
homologs RSW1 and IRX3. Complementation of rsw1 and
irx3 mutants with wild-type RSW1 and IRX3 genes,
respectively, restored the wild-type phenotype, providing
genetic proof of the involvement of these CesA genes in
the biosynthesis and assembly of cellulose micro®brils in
Arabidopsis.
Although similar CesA genes are presumably involved in
the biosynthesis of cellulose in tree xylem (or wood), their
localization and speci®c function have not been documented. It is also unknown whether the same or different CesA
genes are involved in normal and tension wood cellulose
formation in angiosperms. Here we characterize a fulllength CesA cDNA (PtCesA) from aspen (Populus tremuloides) and present in situ evidence that the expression of
this PtCesA gene may be transcriptionally regulated during
both normal and tension wood development.
Results and Discussion
Molecular cloning and comparative sequence analysis of
aspen CesA cDNA suggest that it encodes an authentic
CesA protein
A full-length CesA cDNA from aspen was isolated by
screening a developing xylem cDNA library (Hu et al.,
1998) with an Arabidopsis CesA cDNA as a probe (Wu
et al., 1998). This cDNA clone, designated PtCesA, was
3232 bp long with an open reading frame of 2934 bp
extending from nucleotides 69±3002 (GenBank accession
number AF072131). An ATG at position 69 is in the
optimum context, and is likely to be the initiation codon
based on the criteria of the optimal context sequence and
the longest open reading frame (Joshi, 1987a; Joshi et al.,
1997). A putative polyadenylation signal (AATACA) occurs
16 bp upstream of the polyadenylation site, satisfying the
Figure 1. Domain structure of PtCesA protein showing various conserved regions.
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 495±502
Developing xylem-speci®c aspen cellulose synthase
acid sequence in CR-P-1 near the N-terminus of the PtCesA
protein (Figure 1) hosts the putative zinc-binding motif
encompassing eight highly conserved cysteine residues in
four pairs of CX2C. This zinc-binding motif is considered
important to protein±protein interactions during the cellulose biosynthesis process (Arioli et al., 1998).
These sequence characteristics are typical of plant
cellulose synthases, and are evidence that PtCesA cDNA
encodes a CesA protein for cellulose biosynthesis in
aspen. The high similarity between GhCesA (Pear et al.,
1996) and PtCesA may further suggest that PtCesA is
speci®cally involved during cellulose biosynthesis in the
secondary cell walls.
Southern blot analysis of PtCesA gene family in aspen
genome and tissue-speci®c expression of PtCesA
transcripts by Northern blot and in situ hybridization
analyses
High- and low-stringency Southern blot analysis, using a
5¢-end fragment from PtCesA cDNA as a probe, showed
that aspen possesses a small family of PtCesA genes
(Figure 2a,b). However, upon longer exposure the
Southern blot exhibited multiple weak hybridizing bands
(data not shown), suggesting that a large number of CesA
genes, including PtCesA, may be present in aspen. When a
5¢-end fragment from Arabidopsis RSW1 cDNA (Wu et al.,
1998) was used as a probe, Southern blot analysis revealed
a different band pattern with, in some cases, more than 10
hybridizing bands (Figure 2c). Therefore the PtCesA genespeci®c probe does not cross-hybridize with RSW1-like
genes which may constitute a distinct subset of CesA
genes in aspen. This is consistent with the ®nding (based
on the results from genomic Southern analyses, largescale genome sequencing and EST studies) that most
plant genomes may contain about 10 or more distinct
CesA or CesA-like genes (Cutler and Somerville, 1997;
Delmer, 1999).
Northern blot analysis of RNAs from young aspen stems
(Figure 2d) revealed the near absence of PtCesA transcripts
in internodes undergoing primary growth (internodes 1±4).
The appearance of PtCesA transcripts in internodes 5±10
coincided with the onset of secondary growth. Re-probing
the Northern blot with the 5¢-end fragment of RSW1 cDNA
showed, in contrast, a substantial expression in both
primary and secondary growth internodes (Figure 2e).
These results suggest that primary cell-wall related CesA
members, such as those similar to RSW1, are expressed in
both primary and secondary growth tissues, and that
PtCesA is more associated with secondary growth. The
weak Northern signals in aspen internodes undergoing
primary growth may also suggest that PtCesA gene
expression is speci®c to xylem, of which there is little in
that tissue. Indeed, in situ localization of PtCesA transcripts
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 495±502
497
along the developmental gradient de®ned by stem primary
and secondary growth revealed that the PtCesA gene is
expressed in primary xylem (PX) cells during primary
growth (Figure 3a,b). At this stage the young internodes
are elongating, resulting in the thickening of the primary
xylem cells through the formation of the secondary wall
(Esau, 1967). Thus the concurrence of shoot elongation with
expression of PtCesA suggests an association of the PtCesA
protein with cellulose biosynthesis during secondary cellwall formation. In the later stages of primary growth
(internode 4) an orderly alignment of the primary xylem
cells begins to develop, accompanied by strong expression
of the PtCesA gene in primary metaxylem cells (Figure 3b).
This marks active cellulose biosynthesis in newly formed
tracheary elements. At the beginning of secondary growth
in older internodes (6), expression of the PtCesA gene was
limited to developing secondary xylem (SX) cells (Figure
3c). Instead of elongation, stem radial expansion at this
stage is the main growth development driven by the
thickening in secondary cell walls of the secondary xylem.
Localized expression of PtCesA in the developing secondary xylem cells (SX, Figure 3c) at this stage is again
consistent with the hypothesis that PtCesA encodes a
cellulose synthase that is active in secondary walls of
developing xylem cells. No PtCesA gene expression was
observed in mature primary xylem cells (PX, Figure 3c)
following completion of secondary wall formation. These
results demonstrate xylem-speci®c expression of the
PtCesA gene in cells undergoing cellulose biosynthesis
during secondary cell-wall formation.
The results from Southern, Northern and in situ
hybridization analyses provide evidence that a small
number of CesA genes, the PtCesA homologs, are involved
in the deposition of cellulose in secondary walls of
developing xylem cells in aspen. Repeated screening of
an aspen xylem cDNA library with various plant CesA
gene-related probes always resulted in the isolation of the
same PtCesA cDNA clone. Only one developing xylemassociated CesA-like clone (Al166604), exhibiting a high
(94%) nucleotide sequence identity with PtCesA cDNA,
was found in over 5000 Populus EST clones (Sterkey et al.,
1998). These observations may imply that although
different CesA members might perform different functions
in various cell types (Carpita and Vergara, 1998; Cutler and
Somerville, 1997; Delmer, 1999), an ef®cient cellulose
biosynthesis machinery requiring a few PtCesA-like
synthases is devoted to streamlining the production of
cellulose in tree xylem (or wood).
Heterologous PtCesA gene promoter±GUS fusion
analysis
In order to analyze the regulation of PtCesA gene
expression, its 5¢-¯anking sequence was cloned and
498 Luguang Wu et al.
the PtCesA gene in aspen stems. Developing phloem
®bers, which are also active in cellulose and lignin
biosynthesis (Figure 4d,e), did not show any GUS expression suggesting that the PtCesA gene is not associated
with cellulose biosynthesis in tissue types other than
xylem. The results from promoter activity characterization
(Figure 4), together with those from in vivo expression of
the PtCesA gene (Figures 2 and 3), further support the idea
that the expression of PtCesA is associated with the
biosynthesis of cellulose in secondary walls of developing
xylem cells in aspen. The degree of polymerization and
crystallinity of cellulose is higher in secondary than in
primary cell walls (Haigler and Blanton, 1996; Haigler,
1985; Taylor et al., 1999; Timell, 1986). PtCesA may therefore be involved in a stress-response mechanism of
angiosperms that results in deposition of highly crystalline
cellulose in tension wood. To test whether PtCesA gene
expression is associated with tension wood formation, we
analyzed PtCesA gene promoter activity in transgenic
tobacco under tension stress.
PtCesA gene promoter activity under tension stress
Figure 2. Southern blot analysis of aspen genomic DNA and Northern
blot analysis of aspen RNAs from various tissues.
For Southern blot analysis, genomic DNA (15 mg per lane) was digested
with restriction enzymes PstI (lane P), HindIII (lane H) and EcoRI (lane E),
respectively, and probed with a 32P-labeled 1 kb 5¢-end of PtCesA cDNA
fragment at low (a) and high (b) stringency and with a 32P-labeled 1 kb 5¢
end of Arabidopsis RSW1 cDNA at low stringency (c). Molecular size of
fragments (kb) is shown on the left. Northern blot analysis of total RNA
(30 mg per lane) from aspen stem internodes, probed with a 32P-labeled 5¢
1 kb aspen PtCesA cDNA (d) and a 32P-labeled 5¢ 1 kb Arabidopsis RSW1
cDNA (e) fragments as the probes. Lanes 1, 1st and 2nd internodes; 2,
3rd and 4th internodes; 3, 5th and 6th internodes; 4, 9th and 10th
internodes. Molecular size of transcripts is shown on the right.
characterized. The genomic fragment, designated as
PtCesAP (AF197911), contained 772 bp of promoter sequence, 68 bp 5¢-untranslated leader region and 170 bp
PtCesA coding sequence. To investigate the regulation of
PtCesA expression at the cellular level, PtCesAP-driven
GUS gene expression in transgenic tobacco was then
conducted. In 11 independent transgenic lines, GUS
staining was detected exclusively in xylem tissues of the
stem (Figure 4), root, leaf, ¯ower and fruit (data not
shown), indicating that PtCesAP would direct PtCesA gene
expression in a xylem-speci®c manner that is independent
of the organ. In young stem tissue (internode 3), strong
GUS activity was localized to xylem cells undergoing
primary growth (Figure 4a). However, GUS expression
became localized to developing secondary xylem cells
during secondary growth in internodes 5, 7 and 8 (Figure
4b±d,f), consistent with the in vivo expression pattern of
The stems of several PtCesAP±GUS construct-expressing
plants were bent to create tension stress for various time
intervals (from 4 to 40 h). Surprisingly, tension stress
rapidly induced phloem-speci®c GUS expression (Figure
5), but did not cause an apparent change in GUS
expression in the developing xylem (Figure 5b). Thus
under a normal developmental program of xylem differentiation, PtCesA expression is speci®cally associated with
cellulose biosynthesis in xylem cells as concluded above
(Figure 4), but becomes inducible for the synthesis of
cellulose in phloem ®bers in response to tension stress
(Figure 5). This is consistent with tension wood formation
in which not only xylem, but also phloem ®bers, produce
cellulose with high crystallinity to provide additional
tensile strength to counteract the mechanical stress of
bending (Timell, 1986). After 20 h of bending, GUS
expression was observed in xylem and phloem ®bers in
the stretched portion of the stem experiencing tension
stress. However, GUS expression was turned off in the
compressed tissues on the opposite side of the tension
force (Figure 5c). When tension was applied for 40 h, GUS
expression became restricted to a narrow region on the
upper side of the stem where tension stress was maximum
(Figure 5d), while remaining suppressed in the stemcompression zone (Figure 5d). These observations suggest
that tension stress in angiosperm trees induces a marked
metabolic change involving tension zone-localized cellulose biosynthesis. However, results for model herbaceous
species need to be con®rmed in trees.
Although the molecular basis for tension wood formation is not known, it is hypothesized that an auxin gradient
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 495±502
Developing xylem-speci®c aspen cellulose synthase
499
Figure 3. In situ localization of PtCesA gene
transcripts.
Transverse sections (10 mm) from the 2nd
(a), 4th (b) and 6th (c) aspen stem
internodes were hybridized with DIG-labeled
PtCesA antisense probes, and transverse
sections from the 5th internode with a DIGlabeled PtCesA sense probe as a control (d).
Positive DNA/RNA hybridization signals
stained in purple blue color. Arrows indicate
the
cellular
localization
of
PtCesA
transcripts. No hybridization signal was
detectable in control section (d). PX,
primary xylem cells; SX, secondary xylem
cells. Bar = 100 mm.
Figure 4.
Histochemical
analysis
of
transgenic
tobacco
for
GUS
gene
expression driven by aspen PtCesA gene
promoter, PtCesAP.
Stem transverse sections from the 3rd (a),
5th (b), 7th (c) and 8th (d,f) internodes were
stained for GUS activity. Fluorescence
microscopy (e) shows the same section as
in (d). Lignin auto¯uorescence (blue) was
visualized following UV irradiation at
365 nm. An entire cross-section from the 8th
internode stained for GUS activity is shown
in (f). Bar = 100 mm (a±e); 1.5 mm (f).
between the upper and lower sides of the leaning stem is
associated with increased deposition of highly crystalline
cellulose in cell walls to provide more tensile strength to
counteract gravitational forces acting on the leaning stem
(Timell, 1986). The interactions between auxin gradients,
tension zone-localized factors and the regulatory sequences in the PtCesA gene promoter may connect the
expression of PtCesA in phloem cells with tension stressregulated distribution of carbon for primary (cellulose) and
secondary (lignin) metabolism. During tension cellulose
becomes the major carbon sink in the tension zone, and in
the same zone lignin biosynthesis would be expected to be
attenuated due to the shift in carbon ¯ux towards cellulose
biosynthesis. Thus tension wood in angiosperm trees
always features increased cellulose and decreased lignin
contents (Timell, 1969, Timell, 1986). It may be particularly
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 495±502
important for the long-term structural integrity of trees to
be able to alternate distribution of carbon that is targeted
from structural uses between these two major carbon
sinks (Hu et al., 1999; Li et al., 2000). Our results further
suggest that, in the compression zone lying opposite to the
tension zone, certain auxin imbalance-induced factors may
inhibit PtCesA expression and therefore interrupt cellulose
biosynthesis. Thus lignin would become the major carbon
sink in the compression zone to increase cell-wall compression strength, consistent with the fact that increased
lignin and decreased cellulose contents are normally
found in compression-stressed tissues (Schwerin, 1958;
Timell, 1969, Timell, 1986). Our results also indicate that
the tension stress regulation appears to coordinate the
cellulose-destined carbon distribution with a constant
PtCesA expression in both phloem and xylem cells, as
500 Luguang Wu et al.
Figure 5. GUS expression driven by aspen PtCesA gene promoter in transgenic tobacco plants under tension stress.
Tension stress was induced by bending the transgenic plants for various time periods. Blue arrows indicate the bending sites. Sections taken before
bending (a), and 4 (b), 20 (c), 40 (d) h after bending, respectively, are shown and stained for GUS activity. Longitudinal sections are also shown below
tangential sections (a,c). After 4 (b), 20 (c) and 40 (d) h bending, GUS expression was detected in the developing phloem (Pf) and xylem (Dx) ®bers only on
the upper side of the bent stem. GUS expression in Pf is shown in tangential sections (b) and (d), and in the longitudinal section in inset (b). After 40 h
bending (d) GUS expression was observed only in a narrow region where tension stress was present (inset, d).
indicated by the persistent GUS expression in the tension
zone (Figure 5). This metabolic coordination may be
designed to allow an ef®cient cellulose biosynthetic
system to assemble cellulose micro®brils with high
crystallinity in tissues experiencing tension stress. The
disruption of such a system due to the suppressed PtCesA
expression and ligni®cation-bound carbon ¯ux may then
account for the abnormally low crystallinity of compression cellulose as compared to that of tension cellulose
(Tanaka et al., 1981; Timell, 1986).
The results of our gene regulation and in vivo gene
expression analyses point to a unique role for PtCesA in
cellulose biosynthesis during both normal and abnormal
wood development in aspen. Our molecular evidence
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 495±502
Developing xylem-speci®c aspen cellulose synthase
further suggests that the on/off PtCesA-like gene expression may be part of a signaling mechanism triggering a
stress-related compensatory deposition of cellulose and
lignin that is crucial to growth and development in trees.
Transcriptional control of PtCesA-like gene expression may
be an important regulator of cellulose and lignin deposition
in trees, thus our current research should be broadened to
consider the mechanisms coordinating the biosynthesis of
cellulose and lignin, the two major cell-wall components.
Experimental procedures
Plant materials
Young stem internodes and leaves of aspen (Populus tremuloides
Michx., genotype W271) were collected from greenhouse-grown
4-month-old vegetatively propagated cuttings. Developing secondary xylem was collected as described (Hu et al., 1998). These
plant materials were immediately frozen and stored in liquid
nitrogen until used for isolating DNA and RNA.
Molecular cloning of PtCesA cDNA and gene
A randomly primed 32P-labeled 1651 bp long EcoRI fragment
located in the central UDP-glucose binding region of an
Arabidopsis CesA cDNA (Wu et al., 1998) was used as a probe to
screen about 500 000 plaques of an aspen developing xylem
cDNA library (Hu et al., 1998). Four positive clones were obtained
after three rounds of plaque puri®cation and hybridization.
Automated DNA sequencing from the 3¢ ends of the positive
clones (ABI310 Genetic Analyzer, Perkin Elmer Applied
Biosystems) and analysis of the DNA sequence data using the
GCG software package indicated that these four clones were
identical at the 3¢ end. The longest clone obtained by repeated
screening of the same cDNA library was fully sequenced from
both directions and found to be a full-length cDNA, and was
designated PtCesA.
A 1200 bp long 5¢ fragment of PtCesA cDNA was used to screen
an aspen genomic library constructed by cloning the Sau3AI
partially digested and sucrose gradient-selected genomic DNA
fragments into the BamHI site of a lambda DASH II vector
(ClonTech) (Hu et al., 1998). Five positive clones were obtained
after screening of about 150 000 plaques from the genomic
library, and lambda DNA was isolated using Wizard Lambda
Preps (Promega). An approximately 1 kb 5¢-¯anking region of the
PtCesA open reading frame, designated PtCesAP, was subcloned
into a pCR 2.1 vector (Invitrogen) for DNA sequencing from both
directions.
DNA and RNA gel blot analysis
Total genomic DNA was isolated from young aspen leaves
according to Hu et al. (1998). Aliquots of 15 mg DNA were digested
with restriction enzymes EcoRI, HindIII and PstI individually at
37°C overnight, fractionated on 0.8% agarose gel and blotted onto
a MagnaGraph nylon membrane (Micron Separation Inc).
Southern blot hybridization was performed at either low- (48°C)
or high- (65°C) stringency conditions as described (Hu et al., 1998).
Total RNA was isolated from various aspen stem internodes
according to Hu et al. (1998). Thirty mg total RNA per lane was
fractionated by 2.2 M formaldehyde and 1.0% agarose gel
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 495±502
501
electrophoresis, and blotted onto a Hybond nylon membrane
(Amersham Pharmacia BioTech). The blots were hybridized with
randomly primed 32P-labeled probes from a 5¢ gene-speci®c
region of PtCesA or from the Arabidopsis RSW1 cDNA as
previously (Hu et al., 1998).
Preparation of promoter±GUS fusion constructs and
transformation of tobacco
For the construction of the PtCesAP±GUS fusion binary vector, the
PtCesAP sequence was subcloned into pCR 2.1 vector (Invitrogen)
at the EcoRI site and was subcloned at the HindIII±XbaI sites of
pBI121 vector (ClonTech) in the same reading frame as the GUS
gene. The plasmids were mobilized into Agrobacterium tumefaciens strain C58/pMP90 by the freeze±thaw method. Leaf disc
transformation of tobacco (Nicotiana tabacum cv. Havana) and
histochemical localization of GUS activity was conducted as
described (Hu et al., 1998).
Tension stress treatments
The stems of several transgenic tobacco plants (10±15 cm height)
carrying PtCesAP±GUS constructs were bent to induce tension
stress for 4±40 h. The bent portion (2±3 cm) of the stem was
harvested at the selected time points, and hand sections
(transverse and longitudinal) were used for histochemical staining for GUS activity as described above. Lignin distribution in
plant tissues, as indicated by lignin auto¯uorescence following
UV irradiation at 365 nm, was visualized and photographed using
a Nikon Eclipse 400 ¯uorescence microscope.
In situ hybridization
PtCesA transcripts were detected in young aspen stem sections
by in situ hybridization with transcripts from the highly variable 5¢
region of PtCesA cDNA (PstI and SacI fragment of 771 bp). This
cDNA was subcloned into the plasmid vector pGEMq-3Zf (+)
(Promega) for the production of antisense and sense digoxygenin-labeled transcripts by using T7 and SP6 RNA polymerases (DIG
system, Boehringer Mannheim), respectively. These transcripts
were subjected to mild alkaline hydrolysis in 100 mM NaHCO3
pH 10.2 at 60°C to produce RNA fragments of approximately
200 bp in length (Cox et al., 1984) and used as probes for in situ
localization of mRNA according to a modi®ed procedure of
Jackson (1991). Brie¯y, segments (approximately 5 mm) of aspen
stem were ®xed in 4% (w/v) paraformaldehyde in 100 mM
phosphate buffer (pH 7.0) at 4°C overnight, dehydrated through
an ethanol series, and embedded in paraf®n. Sections (10 mm)
were mounted on Superfrost/plus (Fisher) slides at 42°C overnight, and were followed by dewaxing and rehydration through a
descending ethanol series. The sections were incubated with
proteinase K (10 mg ml±1 in 100 mM Tris±HCl, 50 mM EDTA pH 7.5)
for 30 min, post-®xed with FAA, and acetylated with 0.33% (v/v)
acetic anhydride in 0.1 M triethanolamine-HCl (pH 8.0) prior to
hybridization. The sections were then incubated in the hybridization mixture (approximately 2 mg ml±1 DIG-labeled probes, 50% (v/
v) formamide, 2 3 SSPE, 10% (w/v) dextran sulfate, 125 mg ml±1
tRNA pH 7.5) at 45°C for 12±16 h. Unhybridized single-stranded
RNA probe was removed by treatment with 20 mg ml±1 RNase A in
TE buffer with 500 mM NaCl. The sections were washed twice with
2 3 SSC buffer containing 50% formamide (v/v) for 15 min at 50°C.
Hybridized DIG-labeled probe was detected on sections using
antidigoxygenin antiserum at a 1 : 1500 dilution according to the
manufacturer's protocol (DIG system, Boehringer Mannheim).
502 Luguang Wu et al.
Sections were examined using a Nikon Eclipse 400 light microscope and photographed.
Acknowledgements
We thank Dr Scott Harding and Ms Jacqueline Popko, Michigan
Technological University; Dr Debra Delmer, University of
California, Davis; Dr Candace Haigler, Texas Tech University,
Lubbock and Dr Inder Saxena, University of Texas, Austin for
critical reading of the manuscript and providing many useful
suggestions. This research was supported in part by grants from
the USDA±National Research Initiative Competitive Grants Program (99-35103-7986) and the USDA±McIntire±Stennis Forestry
Research Program.
References
Arioli, T., Peng, L., Betzner, A.S. et al. (1998) Molecular analysis of
cellulose biosynthesis in Arabidopsis. Science, 279, 717±720.
Baskin, T.I., Betzner, A.S., Hoggart, R., Cork, A. and Williamson,
R.E. (1992) Root morphology mutants in Arabidopsis thaliana.
Aust. J. Plant. Physiol. 19, 427±437.
Carpita, N. and Vergara, C. (1998) A recipe for cellulose. Science,
279, 672±673.
Cote, W.A., Day, A.C. and Timell, T.E. (1969) A contribution to the
ultrastructure of tension wood ®bers. Wood Sci. Technol. 3,
257±271.
Cox, K.H., DeLeon, D.V., Angerer, L.M. and Angerer, R.C. (1984)
Detection of mRNAs in sea urchin embryos by in situ
hybridization using asymmetric RNA probes. Dev. Biol. 101,
485±502.
Cutler, S. and Somerville, C. (1997) Cellulose synthesis: cloning in
silico. Current Biol. 7, R108±R111.
Delmer, D.P. (1999) Cellulose biosynthesis: exciting times for a
dif®cult ®eld of study. Ann. Rev. Plant Physiol. Plant Mol. Biol.
50, 245±276.
Delmer, D.P. and Amor, Y. (1995) Cellulose biosynthesis. Plant
Cell, 7, 987±1000.
Esau, K. (1967) The cell wall. In Plant Anatomy, 2nd edn. New
York: Wiley, pp. 33±66.
Haigler, C. (1985) The functions and biogenesis of native cellulose.
In Cellulose Chemistry and Applications (Nevell, T.P. and
Zoronian, S.H., eds). Chichester: Ellis Horwood, pp. 30±83.
Haigler, C. and Blanton, R.L. (1996) New hopes for old dreams:
evidence that plant cellulose synthase genes have ®nally been
cloned. Proc. Natl Acad. Sci. USA, 93, 12082±12085.
Hu, W.J., Kawaoka, A., Tsai, C.J., Lung, J., Osakabe, K., Ebinuma,
H. and Chiang, V.L. (1998) Compartmentalized expression of
two structurally and functionally distinct 4-coumarate: coA
ligase genes in aspen (Populus tremuloides). Proc. Natl Acad.
Sci. USA, 95, 5407±5412.
Hu, W.J., Harding, S.A., Lung, J., Popko, J.L., Ralph, J., Stokke,
D.D., Tsai, C.-J. and Chiang, V.L. (1999) Repression of lignin
biosynthesis promotes cellulose accumulation and growth in
transgenic trees. Nature Biotechnol. 17, 808±812.
Jackson, D. (1991) In situ hybridization in plants. In Molecular
Plant Pathology, A Practical Approach, Vol. 1 (Gurr, S.J.,
McPherson, M.J., Bowles, D.J., eds). Oxford: Oxford
University Press, pp. 163±174.
Joshi, C.P. (1987a) An inspection of the domain between putative
TATA box and translation start site in seventy-nine plant genes.
Nucl Acid Res. 15, 6643±6653.
Joshi, C.P. (1987b) Putative polyadenylation signals in nuclear
genes of higher plants: a compilation and analysis. Nucl. Acids
Res. 15, 9627±9640.
Joshi, C.P., Zhou, H., Huang, X. and Chiang, V.L. (1997) Context
sequences of translation initiation codon in plants. Plant Mol.
Biol. 35, 993±1001.
Li, L., Popko, J.L., Umezawa, T. and Chiang, V.L. (2000) 5Hydroxyconiferyl aldehyde modulates enzymatic methylation
for syringyl monolignol formation, a new view of monolignol
biosynthesis in angiosperms. J. Biol. Chem. 275, 6537±6545.
Matthysse, A.G., White, S. and Lightfoot, R. (1995) Gene required
for cellulose synthesis in Agrobacterium tumefaciens. J.
Bacteriol. 177, 1069±1075.
Norberg, P.H. and Meier, H. (1966) Physical and chemical
properties of the gelatinous layer in tension wood ®bers of
aspen (Populus tremula L.). Holzforchung, 20, 174±178.
Pear, J.R., Kawagoe, Y., Schreckengost, W.E., Delmer, D.P. and
Stalker, D.M. (1996) Higher plants contain homologs of the
bacterial celA genes encoding the catalytic subunit of cellulose
synthase. Proc. Natl Acad. Sci. USA, 93, 12637±12642.
Saxena, I.M., Lin, F.C. and Brown, R.M. (1990) Cloning and
sequencing of the cellulose synthase catalytic subunit gene of
Acetobacter xylinum. Plant Mol. Biol. 15, 673±683.
Saxena, I.M., Brown, R.M., Fevre, M., Geremia, R.A. and
Henrissat, B. (1995) Multidomain architecture of bglycosyltransferases: implications for mechanism of action. J.
Bacteriol. 177, 1419±1424.
Schwerin, G. (1958) The chemistry of reaction wood. Part II. The
polysaccharides of Eucalyptus goniocalyx and Pinus radiata.
Holzforschung, 12, 43±48.
Sterkey, F., Regan, S., Karlson, J. et al. (1998) Gene discovery in
the wood-forming tissues of poplar: analysis of 5692 expressed
sequence tags. Proc. Natl Acad. Sci. USA, 95, 13330±13335.
Tanaka, F., Koshijima, T. and Okamura, K. (1981) Characterization
of cellulose in compression wood and opposite woods of a
Pinus densi¯ora tree grown under the in¯uence of strong wind.
Wood Sci. Technol. 15, 265±273.
Taylor, N.G., Scheible, W.-R., Cutler, S., Somerville, C.R. and
Turner, S.R. (1999) The irregular xylem3 locus of Arabidopsis
encodes a cellulose synthase required for secondary cell wall
synthesis. Plant Cell, 11, 769±779.
Timell, T.E. (1969) The chemical composition of tension wood.
Svensk Papperstidning, 72, 173±181.
Timell, T.E. (1986) Compression Wood in Gymnosperms. Berlin:
Springer Verlag.
Turner, S.R. and Somerville, C.R. (1997) Collapsed xylem
phenotype of Arabidopsis identi®es mutants de®cient in
cellulose deposition in the secondary cell wall. Plant Cell, 9,
689±701.
Wang, H. and Loopstra, C.A. (1998) Cloning and characterization
of a cellulose synthase cDNA (Accession no. AF081534) from
xylem of hybrid poplar (Populus tremula 3 Populus alba). Plant
Physiol. 118, 1101.
Wong, H.C., Fear, A.L., Calhoon, R.D. et al. (1990) Genetic
organization of the cellulose synthase operon in Acetobacter
xylinum. Proc. Natl Acad. Sci. USA, 87, 8130±8134.
Wu, L., Joshi, C.P. and Chiang, V.L. (1998) AraxCelA, a new
member of cellulose synthase gene family from Arabidopsis
thaliana (Accession No. AF062485). Plant Physiol. 117, 1125.
GenBank accession numbers PtCesA (AF072131) and PtCesAP (AF197911).
ã Blackwell Science Ltd, The Plant Journal, (2000), 22, 495±502