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First published online December 30, 2003; 10.1104/pp.103.032904 Plant Physiology 134:224-236 (2004) © 2004 American Society of Plant Biologists
The CesA Gene Family of Barley. Quantitative Analysis of Transcripts Reveals Two Groups of Co-Expressed Genes1School of Agriculture and Wine, and the Australian Centre for Plant Functional Genomics, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia
Sequence data from cDNA and genomic clones, coupled with analyses of expressed sequence tag databases, indicate that the CesA (cellulose synthase) gene family from barley (Hordeum vulgare) has at least eight members, which are distributed across the genome. Quantitative polymerase chain reaction has been used to determine the relative abundance of mRNA transcripts for individual HvCesA genes in vegetative and floral tissues, at different stages of development. To ensure accurate expression profiling, geometric averaging of multiple internal control gene transcripts has been applied for the normalization of transcript abundance. Total HvCesA mRNA levels are highest in coleoptiles, roots, and stems and much lower in floral tissues, early developing grain, and in the elongation zone of leaves. In most tissues, HvCesA1, HvCesA2, and HvCesA6 predominate, and their relative abundance is very similar; these genes appear to be coordinately transcribed. A second group, comprising HvCesA4, HvCesA7, and HvCesA8, also appears to be coordinately transcribed, most obviously in maturing stem and root tissues. The HvCesA3 expression pattern does not fall into either of these two groups, and HvCesA5 transcript levels are extremely low in all tissues. Thus, the HvCesA genes fall into two general groups of three genes with respect to mRNA abundance, and the co-expression of the groups identifies their products as candidates for the rosettes that are involved in cellulose biosynthesis at the plasma membrane. Phylogenetic analysis allows the two groups of genes to be linked with orthologous Arabidopsis CesA genes that have been implicated in primary and secondary wall synthesis.
Cellulose biosynthesis in vascular plants is effected at the plasma membrane by a rosette terminal complex of proteins that contains catalytic cellulose synthase subunits (Roelofsen, 1958  ; Mueller and Brown, 1980; Kimura et al., 1999) and, in all likelihood, ancillary proteins or enzymes required for the extrusion of cellulosic chains and assembly of microfibrils (Doblin et al., 2002). In the single most convincing demonstration of high-level in vitro cellulose biosynthesis by plant enzymes, the rosette complexes can been seen at the termini of cellulose microfibrils synthesized in vitro by membrane extracts of suspension-cultured cells of Rubus fruticosus (Lai-Kee-Him et al., 2002). Although biochemical approaches to the purification and characterization of plant cellulose synthases have met with little success (Delmer, 1999), mutational genetics, gene silencing, and herbicide studies are now providing overwhelming evidence that the catalytic subunits of rosettes are encoded by CesA (cellulose synthase) genes (Pear et al., 1996; Arioli et al., 1998; Burton et al., 2000; Scheible et al., 2001).
Genome sequencing programs and the generation of extensive expressed sequence tag (EST) databases have shown further that plant CesA genes are members of multigene families. There are at least 10 CesA genes in Arabidopsis, 12 in rice (Oryza sativa; Richmond and Somerville, 2000
Additional specialized roles for members of the CesA gene family might include the synthesis of wall polysaccharides other than cellulose. Given that the backbone structures of non-cellulosic wall components such as heteroxylans, xyloglucans, mannans, and (1
Here, the CesA gene family from barley (Hordeum vulgare) has been examined, through cloned cDNAs, EST sequence analysis, and genomic clones, and the locations of the genes on high-density genetic maps have been defined. Quantitative-PCR (Q-PCR; Fink et al., 1998
Cloning the HvCesA cDNAs and Genes A PCR product was initially amplified from a young barley leaf cDNA preparation with degenerate primers from conserved regions of plant CesA genes. This generated a cDNA, designated HvCesA1, which was used to screen a barley suspension-cultured cell cDNA library at low stringency, to yield corresponding fragments of the HvCesA2 and HvCesA3 genes. The HvCesA4 cDNA was first isolated from a 3-d coleoptile library during EST sequencing carried out by Dr. Andreas Graner (Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany). Contiguous sequences for HvCesA5 and HvCesA6 were initially constructed from ESTs listed on the http://cellwall.stanford.edu/Web site, and HvCesA8 was constructed by bridging two singletons listed on the same Web site. The sequences of HvCesA1, HvCesA2, and HvCesA3 were extended through EST sequences from the web site. A 3'-untranslated region (UTR) of HvCesA7, which is 96% identical with HvCesA5, was obtained during Q-PCR experiments. Barley bacterial artificial chromosome (BAC) clones corresponding to most of the individual HvCesAs were also identified as follows: HvCesA1, 337H09; HvCesA2, 69M22; HvCesA3, 283N14; HvCesA4, 45J23; HvCesA5/7, 627G13; and HvCesA6, 453O07. Sequences from the BAC clones were used to extend 5' sequences of the cDNAs. Once the sequences were assembled, PCR was used to generate near full-length (3.6-3.9 kb) single cDNA fragments corresponding to each of the HvCesA1, HvCesA2, and HvCesA6 genes. The other cDNAs were truncated at their 5' ends by between 30 bp and about 1.8 kb because the corresponding BAC clones did not contain the 5' regions of the genes. The respective sizes of cDNAs for HvCesA1, HvCesA2, HvCesA3, HvCesA4, HvCesA5/7, HvCesA6, and HvCesA8 were 3,614, 3,910, 3,180, 1,814, 2,769, 3,739, and 1,246 bp. All have open reading frames that encode polypeptides of 1,000 to 1,100 amino acid residues. Their sequences have been submitted to the databases under accession numbers AY483150, AY483152, AY483151, AY483154, AY483153, AY483155, and AY483156, respectively.
The sequences of the barley cDNAs are reconciled with available EST sequences in Figure 1, where most ESTs in the public databases can be assigned to the genes cloned here. On this basis, it is concluded that the barley CesA gene family has at least eight members. Several singleton EST sequences are currently unassigned, and, although sequence data for ESTs are not always accurate, it is possible that these could represent additional HvCesA genes.
The HvCesA genes were mapped using several mapping populations and their positions in relation to DNA molecular markers are shown in Figure 2. The precise map positions of the barley genes have been lodged on the GrainGenes Web page under the CesA designation (http://wheat.pw.usda.gov/index.shtml). No polymorphisms for the partial HvCesA4 cDNA were detected in any of the parental lines, so this gene could not be mapped to a specific locus, and the HvCesA8 gene has not yet been mapped. However, the HvCesA4 gene has been mapped to the long arm of chromosome 1H, using wheat barley addition lines (Islam et al., 1981
An unrooted, radial phylogenetic tree of the barley HvCesA gene family was generated with the software program ClustalX (Thompson et al., 1997
The relative levels of individual HvCesA mRNAs were determined using real-time Q-PCR (Fink et al., 1998
The NF, derived from the geometric mean of expression data for the most stably expressed control genes by the geNorm program (Vandesompele et al., 2002 In the present work, NF values for most cDNA samples remained relatively unchanged between five and two control genes, except that the NFs for the root tip sample decreased significantly between the use of five and two control genes (Fig. 4). The order of elimination of control genes was HvCesA1, HvHSP70, and HvTub, leaving HvCycl and HvGAPDH. It can be seen in Figure 4 that the elimination of HvTub to leave just two control genes significantly decreased the NF value for the root tip cDNA. The addition of HvHSP70 markedly increased the root tip NF value and at the same time introduced a gene with expression that varies more widely across the tissue series, that is, it has a higher M value than the best three control genes. On this basis, the NF values based on the control genes HvTub, HvCycl, and HvGAPDH were used for normalization of the expression data of the HvCesA gene family (Table II). Normalized expression data from the HvCesA genes were calculated by dividing the raw data by the NF value. SDs were calculated from four replicate PCR experiments per cDNA sample.
Data from the tissue series were used to determine the most appropriate genes for the tissue subseries comprising seedling root, seedling leaf, coleoptile, and mature leaf. When the data were considered for the pair of tissues from leaf or root, the order of fitness of control genes was different from that calculated from all the tissues in the series. This is not surprising because the tissues are metabolically quite distinct (Vandesompele et al., 2002
Overall, HvCesA transcript levels were highest in 3-d coleoptiles, stems taken from the fully extended internode below the spike before anthesis, and the maturation zone of roots (Fig. 5A). Levels were relatively low in developing grain and floral tissues. Closer scrutiny of the data in Figure 5A revealed two distinct groups of apparently co-expressed genes. Group I consisted of HvCesA1, HvCesA2, and HvCesA6. Using the primer sets shown in Table IV, HvCesA1 levels in the majority of tissues were about the same as HvCesA2, and both were consistently 2- to 3-fold higher than HvCesA6 (Fig. 5B). The second group of apparently cotranscribed genes (Group II) consisted of HvCesA4, HvCesA7, and HvCesA8 (Fig. 5C). Within Group II, HvCesA8 transcript levels are consistently about 2-fold higher that HvCesA4 mRNA, whereas HvCesA7 transcript levels are about 10% to 15% of HvCesA8 levels (Fig. 5C). Transcripts of HvCesA5 were of lowest abundance in all tissues, at 0.001% to 0.09% of HvCesA1 transcript levels (Fig. 5A), and were detected mainly in mature stem and in the maturation zone of roots. Transcripts of HvCesA3 were generally low, except in leaf tips (Fig. 5A), and expression patterns of this gene appeared to be independent of the two co-expressed groups.
The abundance of individual HvCesA mRNAs in various growth zones of vegetative tissues was examined in segments of young seedling leaves (7 d old), young roots (5 d old) and coleoptiles (5 d old), as indicated in Figure 6, and in mature leaves (fifth leaf). In young seedling leaves, levels of most mRNAs were highest in the cell elongation zone above the leaf base (Figs. 7, A and B). Again, levels of the Group I mRNAs of HvCesA1, HvCesA2 and HvCesA6 were highest and in approximately constant proportions along the seedling leaf (Fig. 7A). Group II mRNAs (HvCesA4, HvCesA7, and HvCesA8) were consistently lower in abundance than the Group I mRNAs but also peaked in the seedling leaf elongation zone (Fig. 7B). However, HvCesA3 levels showed a completely different distribution pattern, reaching maximal levels in region B, just below the leaf tip (Fig. 7C).
After the observation that HvCesA3 mRNA levels were relatively abundant in young leaf tips (Figs. 5A and 7C), transcript profiles were examined in segments of mature leaves (Fig. 8). In mature leaves, HvCesA3 mRNAs were more abundant than those of any other HvCesA and peaked in the middle region of the leaf (Fig. 8).
In root segments, two distinct distribution patterns can be seen (Figs. 9, A and B). The Group I HvCesA1, HvCesA2, and HvCesA6 transcripts were relatively evenly distributed along the root (Fig. 9A), whereas the Group II HvCesA4, HvCesA7, and HvCesA8 transcripts decreased in abundance toward the root tip (Fig. 9B). In these tissue segments, HvCesA3 transcript levels followed the Group II pattern (data not shown).
In coleoptiles, Group I and II transcripts appear to decrease from the base to the tip (Fig. 10, A and B), although the levels of the Group I transcripts (Fig. 10A) are much higher than the Group II transcripts (Fig. 10B). In the coleoptile sections, HvCesA3 transcript levels remained relatively high in all segments (data not shown), in contrast to both Groups I and II, which decreased from the base to the top of the coleoptiles (Fig. 10).
Near full-length cDNAs for HvCesA1, HvCesA2, and HvCesA6 were obtained, together with truncated cDNAs encoding several other HvCesA genes. It is concluded at this stage that there are at least eight CesA genes in barley and that these are transcribed at levels sufficient to detect through cDNA or EST library screening (Fig. 1). In Arabidopsis, there are 10 CesA genes, whereas in rice and maize, there are 12 and at least nine, respectively (Holland et al., 2000 ; Richmond and Somerville, 2000; Dhugga, 2001). The number of barley BAC clones screened in the present work was calculated to cover 90% of the barley genome. Thus, it is possible that additional HvCesA genes are present and that transcripts will be detected in specific tissues not examined here or will have sequences so similar to others that it will not be possible to differentiate between true genetic differences, varietal differences, and sequencing errors. All of the HvCesA genes that covered the putative catalytic region of the barley enzymes encode proteins with a D,D,D,QVRRW motif; this is characteristic of plant cellulose synthases (Pear et al., 1996). Molecular mapping studies showed that the HvCesA genes are scattered across the barley genome (Fig. 2), as observed for the Arabidopsis and maize CesA genes (Holland et al., 2000).
Q-PCR, in which geometric averaging of multiple internal control gene transcripts is important for the normalization of transcript abundance (Vandesompele et al., 2002 The other two members of the barley HvCesA gene family did not follow Group I or Group II transcription patterns and, therefore, appeared to be independently regulated. Levels of HvCesA5 mRNA were extremely low in all tissues examined. However, HvCesA3 mRNAs were more abundant, particularly toward the tips of young and mature leaves, where HvCesA3 was the most abundant CesA mRNA by far (Fig. 5). Thus, HvCesA3 may play a specific or specialized role in cellulose synthesis in these regions of barley leaves or may be involved in the synthesis of wall polysaccharides other than cellulose.
It can be concluded from these data that Q-PCR is sufficiently sensitive and precise to confidently detect co-expression of groups of genes through constant relative proportions of their mRNAs (Figs. 5 and 7, 8, 9, 10), provided that transcript levels are carefully normalized (Fig. 4; Vandesompele et al., 2002
The co-expression patterns revealed by the Q-PCR in a wide range of barley tissues are suggestive of functional links between individual HvCesA genes. The three Group I HvCesA1, HvCesA2, and HvCesA6 transcripts are most abundant in tissues and tissue segments where primary cell wall synthesis would be expected to predominate (Figs. 5 and 7, 8, 9). It is possible that the protein products of the three genes are required for the formation of a single cellulose-synthesizing complex in rosettes of the plasma membrane during cell wall synthesis. In the model for rosette structure presented by Doblin et al. (2002
Similarly, the three Group II HvCesA4, HvCesA7, and HvCesA8 genes appear to be co-expressed in tissues in which secondary wall synthesis would be proceeding, including stems and the maturation zones of roots (Fig. 5A). Consistent with this suggestion, Taylor et al. (2003
Williamson et al. (2002
In contrast, semiquantitative RT-PCR of ZmCesA mRNA levels in maize and OsCesA mRNAs in rice did not reveal any obvious groups of genes with similar relative concentrations in different tissues (Holland et al., 2000
The emerging evidence that groups of three different CesA proteins might be required for cellulose biosynthesis, in both primary and secondary walls, and in both monocotyledons and dicotyledons (Figs. 5 and 7, 8, 9, 10; Eckardt, 2003 Despite these similarities, it must be clearly stated at this stage that the roles of the barley Group I and Group II CesA proteins in primary and/or secondary wall synthesis remain to be demonstrated unequivocally. We have tried tissue printing and in situ PCR procedures in attempts to correlate Group I and Group II transcripts with primary and secondary wall formation (data not shown) but have been unable to convincingly show that specific transcripts are more abundant in cells synthesizing the two different wall types. It is likely that higher resolution immunogold labeling of specific HvCesA proteins will be necessary to define the precise cellular location of individual cellulose synthases in various tissues at different stages of growth and development. In the absence of well-characterized barley mutant libraries, it also will be necessary to analyze the functions of individual HvCesA genes through specific gene silencing, both transiently and in stably transformed plants.
Plant Material
Barley (Hordeum vulgare L. cv Sloop) plants were grown in a greenhouse under a day/night temperature regime of 23°C/15°C. For non-greenhouse-grown samples, grain was germinated either in damp vermiculite or on damp paper towels in the dark for 3 to 6 d at 20°C. At harvest, seedling leaves from vermiculite grown grain were about 75% of their final length. Seedling leaves of about 13 cm in length were used to isolate leaf tip (the top 7 mm of the leaf) and 3 mm at the base (cell division zone; Fig. 5). In addition, seedling leaf blades were divided into five sections, designated sections A (leaf tip) to E (leaf base; Fig. 6). Segment E would contain dividing and some elongating cells; at segment D elongation would be complete and secondary wall synthesis would be under way; and in segments C and B, there would be no growth but wall maturation would be occurring (Schünmann et al., 1997 Similarly, root tip (1 cm, containing root cap, meristem, and elongation zone) and mature root (1-cm section about 6 cm behind the root tip, containing the differentiation and maturation zone) were harvested (Fig. 5). Selected 1-cm sections of roots harvested after 5 d were also excised (Fig. 6). In the root tip, cell elongation would be occurring; in segment 2, growth would be complete, but secondary wall synthesis would begin; in segment 3, the xylem would be maturing, whereas in segment 4, lateral root formation would be occurring (Fig. 6; B. Atwell, personal communication). Three-day coleoptiles were divided into three sections of about 1 cm each (designated tip, middle, and base; Fig. 6). Cells in the basal region were expanding, those in the middle segment were fully expanded, and those in the tip were generally shorter; secondary wall formation would be restricted to the two small vascular bundles. Floral tissues, consisting of anthers and pistil, were collected about 2 weeks before anthesis and at anthesis. Stem tissue was taken from the upper internode, below the pre-anthesis spike (i.e. below the peduncle); cell elongation would have ceased in this segment. Developing grain was collected 3 and 13 DPA.
A barley cDNA library derived from RNA of suspension-cultured cell lines was prepared in Full or partial cDNAs in one contiguous segment were obtained by PCR using the Elongase Taq polymerase (Invitrogen, Carlsbad, CA) and primers designed to the 5'- and 3'-UTRs of the HvCesA cDNAs, using various cDNA populations prepared from the tissues listed above as templates. The cDNAs were cloned into the pGEM-T Easy vector (Promega, Madison, WI), and both strands were sequenced using Big Dye 3 chemistry on an ABI 3700 (Applied Biosystems, Foster City, CA) capillary sequencer.
The barley doubled haploid mapping populations Chebec x Harrington (120 lines), Galleon x Haruna Nijo (112 lines), and Clipper x Sahara (150 lines) were used to map the HvCesA genes (Karakousis et al., 2003
Multiple sequence alignments and an unrooted, radial phylogenetic tree of the barley HvCesA gene family were generated using ClustalX (Thompson et al., 1997
Total RNA was extracted from at least three individual samples of all tissues, using a commercially prepared guanidine reagent, TRIzol (Invitrogen) according to the manufacturer's instructions. Purified RNA was treated with DNaseI using the DNA-free kit (Ambion, Austin, TX) according to the manufacturer's instructions. RNA integrity was checked on a 1.6% (w/v) agarose gel containing ethidium bromide. For cDNA synthesis, four independent reactions were undertaken for each tissue and the products pooled. Thus, 2 µg of each RNA was mixed with 1 µL of 50 nM oligo-dT(12-20) primer, 1 µL of 10 mM dNTP mix, and sterile water to a volume of 10 µL. The reaction was heated at 65°C for 5 min and snap cooled on ice. A master mix (10 µL) was added to each reaction and contained 2x RT buffer, 0.1 M dithiothreitol, 50 mM MgCl2, 40 units of RNAseOUT (Invitrogen), and 50 units of Superscript II RT. The reaction was incubated at 42°C for 1 h and for 15 min at 75°C. The cDNA was stored at -20°C.
The primer pairs for control genes and specific HvCesA genes were designed for barley var. Sloop and are listed in Table III. Stock solutions of the PCR product were prepared from a cDNA population generated from 3-d-old coleoptile RNA, purified, and quantified by HPLC. The coleoptile-derived cDNA (1 µL) was amplified in a reaction containing 10 µL of QuantiTect SYBR Green PCR reagent (Qiagen, Valencia, CA), 3 µL each of 4 µM forward and reverse primers, and 3 µL of water. The amplification was effected in a RG 2000 Rotor-Gene Real Time Thermal Cycler (Corbett Research, Sydney) as follows: 15 min at 95°C followed by 45 cycles of 20 s at 95°C, 30 s at 55°C, 30 s at 72°C, and 15 s at 80°C. A melt curve was obtained from the PCR product at the end of the amplification by heating from 70°C to 99°C. During the amplification, fluorescence data was acquired at 72°C and 80°C to gauge the abundance of the individual genes in the coleoptile cDNA preparation. From the melt curve, the optimal temperature for data acquisition was determined (Table III).
Between four and six independent 20 µL PCR reaction mixes were combined and purified by HPLC (Wong et al., 2000 A dilution series covering seven orders of magnitude was prepared from the 109 copies µL-1 stock solution to produce solutions covering 107 to 101 µL-1. Three replicates of each of the seven standard concentrations were included with every Q-PCR experiment, together with a minimum of two "no template" controls. For all genes except HvCesA5, a 1:10 dilution of the cDNA was sufficient to produce expression data with an acceptable SD. Undiluted cDNA was required for the determination of HvCesA5 expression levels because of its low abundance. Four replicate PCRs for each of the cDNAs were included in each experiment. For the Q-PCR experiments, 1 µL cDNA solution was used in a reaction containing 10 µL of QuantiTect SYBR Green PCR reagent, 3 µL each of the forward and reverse primers at 4 µM, 0.6 µL 10 x SYBR Green in water (freshly diluted 10,000x in dimethyl sulfoxide), and 2.4 µL of water. Reactions were performed as follows: 15 min at 95°C followed by 45 cycles of 20 s at 95°C, 30 s at 55°C, 30 s at 72°C, and 15 s at the optimal acquisition temperature (Table III). A melt curve was obtained from the product at the end of the amplification by heating from 70°C to 99°C. PCR products were separated by electrophoresis in 2.5% (w/v) agarose-Tris-borate/EDTA-ethidium bromide gels. The Rotor-Gene V4.6 software (Corbett Research) was used to determine the optimal cycle threshold from the dilution series, and the mean expression level and SDs for each set of four replicates for each cDNA were calculated.
We thank Margie Pallotta for undertaking the mapping experiments; Keith Gatford for help with the preparation of plant material; and Brian Atwell, Tony Bacic, Peter Chandler, and Ed Newbigin for valuable advice. Received September 4, 2003; returned for revision September 22, 2003; accepted October 8, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.032904.
1 This work was supported by the Australian Research Council (grant to G.B.F.) and by the Grains Research and Development Corporation (grant to G.B.F.). * Corresponding author; e-mail geoff.fincher{at}adelaide.edu.au; fax 61-8-8303-7109.
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ABSTRACT
RESULTS
3,1
-D-glucans are chemically analogous with cellulose (Fincher and Stone, 1993
-tubulin (HvTub), heat shock protein 70 (HvHSP70), and cyclophilin (HvCycl), were evaluated as control genes for the Q-PCR analyses and for the calculation of reliable NFs used in comparisons between cDNAs from different tissues. For each control gene, gene-specific primers were used and individual genes were selected on the basis of their high-level, stable transcriptional activity in a range of barley tissues (our unpublished data; Haendler et al., 1987
-ZAPII, according to the manufacturer's instructions (Stratagene, La Jolla, CA) and screened with a PCR fragment amplified with degenerate primers designed to match conserved regions in plant CesA genes. Subsequently, unique 3'-UTR probes were generated by PCR and used to screen the barley BAC library (cv Morex) obtained from the Clemson University Genomics Institute (SC; 
