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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. 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