JXB Advance Access originally published online on July 16, 2003
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Journal of Experimental Botany, Vol. 54, No. 390, pp. 2187-2188,
September 1, 2003
© 2003 Oxford University Press
Isolation and characterization of a new, full-length cellulose synthase cDNA, PtrCesA5 from developing xylem of aspen trees
Received 6 February 2003; Accepted 2 June 2003
Plant Biotechnology Research Center, School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI 49931, USA
* To whom correspondence should be addressed. Fax: +1 906 487 2915. E-mail: cpjoshi{at}mtu.edu
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Abstract |
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Cellulose synthases (CesA) represent a group of ß-1,4 glycosyltransferases involved in cellulose biosynthesis. Recent molecular studies confirmed that a large CesA gene family encoding various CesA isoforms exists in plants. The isolation and characterization of a new, full-length CesA cDNA, PtrCesA5 from an economically important tree, quaking aspen (Populus tremuloides) is reported here. The predicted PtrCesA5 protein is highly similar to Arabidopsis AtCesA3 (88%) involved in primary cell wall synthesis. However, gene expression studies here suggest that PtrCesA5 transcripts are greatly enhanced in developing xylem tissues enriched in secondary cell wall synthesis as compared with leaf tissues actively undergoing primary cell wall synthesis. Availability of this new, dual-function CesA from a woody species will assist in a better understanding of the mechanism of cellulose biosynthesis during wood development.
Key words: Aspen, cellulose, CesA, poplar, wood.
A fine balance between the flexibility, rigidity and strength of cell walls determines the ultimate shape and size of plant cells that is important to normal growth and development of plants. Cellulose is a major component of plant cell walls, but the process of cellulose deposition in various types of cell walls is poorly understood (Haigler et al., 2001). At least ten different CesA genes (AtCesA1–AtCesA10) exist in the Arabidopsis genome. Of these, three CesA genes, AtCesA1, AtCesA3 and AtCesA6 are involved in primary cell wall development, whereas three other genes AtCesA4, AtCesA7 and AtCesA8 play roles in secondary cell wall synthesis (Joshi, 2003). Thus at least two major, but distinct, groups of CesAs are associated with cellulose biosynthesis in primary and secondary cell walls in Arabidopsis. Extension of this concept to forest trees may assist in designing better genetic strategies for the improvement of quantity and quality of cellulose in wood. But little is known about the extent of the CesA gene family from trees that provide raw materials for many forest products. So far only three full-length CesA cDNAs, PcCesA1, PtrCesA1 and PtrCesA2 are reported from trees (Wang and Loopstra, 1998; Wu et al., 2000; Samuga and Joshi, 2002). Thus, the identification of new cDNAs from tree genomes is essential for enhancing knowledge of cellulose biosynthesis in trees that has many fundamental and commercial implications. The isolation and characterization of a new full-length CesA cDNA from aspen, PtrCesA5 that is highly similar to Arabidopsis AtCesA3 implicated in primary cell wall formation is reported here.
The PtrCesA5 cDNA (GenBank accession number AY055724
[GenBank]
) was isolated by screening of an aspen developing xylem cDNA library constructed in Lambda Zap II vector (Stratagene) (kindly provided by Drs Laigeng Li and Vincent Chiang) using a partial aspen CesA radioactive probe. This CesA probe was a RT-PCR product selected from a pool of 
600 bp cDNA fragments amplified from xylem RNA by using the degenerate primers (Forward primer: 5'-TGYTATGTYCAGTTYCCWC-3' and Reverse primer: 5'-GANCCRTARATCCAYCC-3') synthesized on the basis of the conserved amino acid sequences flanking the second hypervariable region, HVRII (X Liang and CP Joshi, unpublished data). The full-length PtrCesA5 cDNA is 3532 bp long with a 3234 bp open reading frame encoding a 1078 amino acid protein with a calculated molecular mass of 119 860 Da. All conserved features of typical plant CesA proteins namely a zinc-binding domain, eight transmembrane domains, two hypervariable regions, and processive glycosyltransferases signature motif D-D-D-QXXRW are present in PtrCesA5 protein (Joshi, 2003). Sequence comparison of PtrCesA5 with ten Arabidopsis CesA genes suggested that AtCesA3 (GenBank Accession number AF027174
[GenBank]
) showed a highest identity of 78% at the nucleotide level and an 84/88% identity/similarity at the amino acid level. Moreover, the predicted peptide sequence of PtrCesA5 has a much greater identity with AtCesA3 than with the three other Populus CesAs available namely PcCesA1 (66%), PtrCesA1 (64%), and PtrCesA2 (71%). This confirms the earlier conclusion by Holland et al. (2000) that, in plants, CesA orthologues appear to be more similar than paralogues. Moreover, the first hypervariable region, HVRI of PtrCesA5 is only 38%, 27%, and 46% identical to PcCesA1, PtrCesA1, and PtrCesA2, respectively. Therefore, the corresponding cDNA regions from PtrCesA5 were used as a gene-specific probe for further molecular analysis.
Southern analysis was done by hybridizing EcoRI, HindIII and XbaI restriction enzyme-digested aspen genomic DNA blots at 60 °C with a 32P-labelled probe representing the HVRI region of PtrCesA5. This region lacks EcoRI, HindIII and XbaI target sites. Less than 10% divergence between probe and target was expected. Southern hybridization showed one to two bands in each lane (Fig. 1A) suggesting that PtrCesA5 belongs to a small gene family of one or two genes in the aspen genome.
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Northern hybridization and RT-PCR techniques were used to compare the PtrCesA5 transcript levels in various aspen tissues. A northern blot of total RNA isolated from aspen leaves, shoot apices, developing xylem tissues, and young internodes was hybridized at 60 °C with a 32P-labelled PtrCesA5 HVRI probe. It revealed the presence of higher levels of PtrCesA5 transcripts in developing xylem, but weaker levels in three other tissues that are enriched in primary wall formation (Fig. 1B). Prior to blotting, the RNA gel was stained with ethidium bromide to confirm equal loading. The presence of smaller-size bands than the expected 3.5 kb band in the northern blot may be attributed to presence of degradation products of the 3.5 kb long PtrCesA5 mRNAs. For RT-PCR, PtrCesA5 HVRI region-specific primers (Forward primer 5'-CACCAATGGACAT GAGGTT-3' and Reverse primer 5'ACATCCGTAGCAGCA TCAAT 3') were used along with the internal control, 5.8S rRNA-specific primers, following the procedure described earlier (Samuga and Joshi, 2002). Again, a stronger amplification was observed in developing xylem as compared to that in leaves, shoot apices and young internodes (Fig. 1C). Thus, both these expression analyses confirm a higher-level expression of PtrCesA5 transcripts in developing xylem tissues actively synthesizing secondary cell walls.
A similar RT-PCR experiment in cotton also reported the expression of the GhCesA3 gene both during primary cell wall as well as secondary cell wall biogenesis phases of cotton fibre development (Laosinchai et al., 2000). Their observation agrees with the expression data presented here showing that PtrCesA5 is also highly expressed in developing xylem undergoing significant secondary cell-wall biosynthesis as well as leaf, young internodes and apices undergoing significant primary cell-wall biosynthesis, but at a lower level. Moreover, predicted PtrCesA5 protein shows a higher percentage identity/similarity (89/91%) to cotton GhCesA3 than Arabidopsis AtCesA3 (84/88%). Thus PtrCesA5 may be serving a dual role as a CesA involved in both primary and secondary cell wall biosynthesis. Further studies are currently in progress to substantiate this hypothesis.
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