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Published in final edited form as: J Exp Bot. 2012 Oct;63(16):6045–6056. doi: 10.1093/jxb/ers255

Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A

Gea Guerriero 1,*,†, Oliver Spadiut 2, Christine Kerschbamer 1, Filomena Giorno 1, Sanja Baric 1, Inés Ezcurra 3,†
PMCID: PMC4944836  EMSID: EMS50177  PMID: 23048131

Abstract

Cellulose synthase (CesA) genes constitute a complex multigene family with six major phylogenetic clades in angiosperms. The recently sequenced genome of domestic apple, Malus×domestica, was mined for CesA genes, by blasting full-length cellulose synthase protein (CESA) sequences annotated in the apple genome against protein databases from the plant models Arabidopsis thaliana and Populus trichocarpa. Thirteen genes belonging to the six angiosperm CesA clades and coding for proteins with conserved residues typical of processive glycosyltransferases from family 2 were detected. Based on their phylogenetic relationship to Arabidopsis CESAs, as well as expression patterns, a nomenclature is proposed to facilitate further studies. Examination of their genomic organization revealed that MdCesA8-A is closely linked and co-oriented with WDR53, a gene coding for a WD40 repeat protein. The WDR53 and CesA8 genes display conserved collinearity in dicots and are partially co-expressed in the apple xylem. Interestingly, the presence of a bicistronic WDR53–CesA8A transcript was detected in phytoplasma-infected phloem tissues of apple. The bicistronic transcript contains a spliced intergenic sequence that is predicted to fold into hairpin structures typical of internal ribosome entry sites, suggesting its potential cap-independent translation. Surprisingly, the CesA8A cistron is alternatively spliced and lacks the zinc-binding domain. The possible roles of WDR53 and the alternatively spliced CESA8 variant during cellulose biosynthesis in M.×domestica are discussed.

Keywords: Bicistronic mRNA, cellulose synthase, collinear genes, Malus×domestica, WD40 repeats, zinc motif

Introduction

The plant cell wall, a complex structure of polysaccharides, has attracted a lot of interest in the past few years because of its potential as a source for biofuels (Sanderson, 2011), and its remarkable material properties (Svagan et al., 2007). Cellulose synthases(CESAs; EC2.4.1.12) belong to the glycosyltransferase family 2 (GT2), which also includes chitin and hyaluronan synthases (Coutinho et al., 2003). CESAs were first identified in the Gram-negative bacterium Gluconoacetobacter xylinus (Saxena et al., 1994) and then later found in other organisms, including plants (Arioli et al., 1998), oomycetes (Grenville-Briggs et al., 2008; Fugelstad et al., 2009), and tunicates (Matthysse et al., 2004; Nakashima et al., 2004). Ten CESAs were identified in Arabidospis thaliana (Richmond and Somerville, 2000), 18 in Populus trichocarpa (Djerbi et al., 2005), and other orthologues are being progressively found by mining the increasing number of sequenced genomes. Despite the numerous studies on CESAs, the detailed mechanisms regulating cellulose deposition and crystallization are still not completely understood (Guerriero et al., 2010; Endler and Persson, 2011).

Plant cell walls play an important role in plant physiology: they regulate cell growth, function as a signalling platform (Ellis et al., 2002), and constitute a barrier against water loss and pathogens (Sieber et al., 2000). The presence of a cell wall integrity (CWI) signalling pathway similar to that present in the yeast Saccharomyces cerevisiae (Levin, 2005) has been demonstrated in plants (Hématy et al., 2009; Ringli, 2010; Hamann and Denness, 2011): the cell wall senses perturbations of its own structural and functional integrity, which is usually altered in situations of exogenous stresses (Steinwand and Kieber, 2010). Signalling cascades are then triggered, which activate hormone responses (Zhang et al., 2011), ectopic lignin deposition (Caño-Delgado et al., 2003; Denness et al., 2011), and/or the synthesis of antimicrobial secondary metabolites (Hernández-Blanco et al., 2007).

Recent advances in the accumulation of DNA sequence information from multiple plant species, including fruit trees such as Malus×domestica (Velasco et al., 2010), allow comparative genomics of CesA gene family members, to identify new features such as conserved neighbouring genes. In this study, 13 CesA genes were identified in the economically important fruit tree M.×domestica. It is shown that one of these genes, which was named MdCesA8-A on the basis of the amino acid sequence similarity to A. thaliana CESA8/IRX1 (At4g18780), is adjacent to and co-oriented with WDR53, encoding a WD40 domain protein that is conserved in diverse eukaryotes, and that this collinear arrangement is conserved in dicot species. MdWDR53 and MdCesA8-A are partially co-expressed in the xylem and transcribed as a bicistronic mRNA in phytoplasma-infected phloem. The bicistronic WDR53–CesA8A transcript contains a spliced intergenic spacer (IGS) with a putative internal ribosome entry site (IRES), and an alternatively spliced CesA8A cistron, which lacks the zinc-binding motif. The possible roles of WDR53 and the alternatively spliced CESA8 protein, altCESA8A, in cellulose biosynthesis, during normal development, and in plant–microbe interactions in domesticated apple are proposed and discussed.

Materials and methods

Data mining and bioinformatics analysis

The identification of putative full-length CesA genes from M.×domestica was carried out by performing BLASTp searches of the predicted apple CESA homologues (http://www.rosaceae.org/projects/apple_genome) against non-redundant protein databases of P. trichocarpa and A. thaliana from the National Centre for Biotechnology (NCBI; http://www.ncbi.nlm.nih.gov). Sequence alignments were performed using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Transmembrane domain prediction was performed using TMHMM at http://www.cbs.dtu.dk/services/TMHMM-2.0/. The phylogenetic trees were built by multiple sequence alignment using MUSCLE (Edgar, 2004), and phylogeny was analysed using PhyML (Guindon and Gascuel, 2003). For CESA alignment, the conserved amino acid regions in the catalytic domain between regions U1 and U4 were used (Carroll and Specht, 2011). Sequences of WDR53 were obtained by blasting the NCBI protein database of specific taxa with MdWDR53, and subsequently back-blasting best matches into the database to confirm WDR53 identity. Trees were rendered using TreeDyn (Chevenet et al., 2006).

Plant material

The experimental plants (M.×domestica Borkh. ‘Golden Delicious’ Clone B on M9 rootstock) were obtained as knip-boom trees from a commercial nursery. Plants infected by ‘Candidatus Phytoplasma mali’ were obtained with the chip-budding technique, by using shoots from naturally infected trees showing pronounced disease symptoms. All the infected donor trees used for this study carried phytoplasma subtype AT-2/rpX-A. Control plants were treated using shoots from healthy apple trees. The presence of the pathogen was confirmed by real-time PCR (Baric et al., 2011), and symptoms were evaluated by scoring the presence of ‘witches’ brooms’.

RNA extraction, cDNA synthesis, and real-time PCR from different apple tissues

Total RNA was extracted from 100 mg of apple tissue (leaves, root tips, buds in the ‘green tip’/‘quarter inch green’ stage, phloem, and xylem from branches) from 2-year-old potted apple trees, by using the RNeasy Plant Mini Kit (Qiagen), coupled with on-column DNase I digestion. RNA was retro-transcribed using the Superscript III First Strand cDNA Synthesis kit (Invitrogen). For quantitative real-time PCR analysis, 40 ng of cDNA was amplified using SYBR GreenER qPCR SuperMix Universal (Invitrogen) on a 7500 Fast Real-time PCR System (Applied Biosystems) with the ROX Reference Dye. The reactions were performed in triplicate and repeated on three biological independent replicates. The primers used are reported in Supplementary Tables S1 and Table S2 available at JXB online. The real-time PCR experiments were carried out for 11 CesA genes, since it was not possible to design primers discriminating MdCesA3-D and MdCesA6-C from MdCesA3-C and MdCesA6-D. A dissociation kinetics analysis was performed at the end of the experiment to check the specificity of annealing. Results were analysed with Q-gene software (Muller et al., 2002) and normalized against the housekeeping genes ubiquitin and glyceraldehyde phosphate dehydrogenase (GAPDH; accession nos EB109811 and CN929227, respectively). All PCR products were checked on agarose gels.

Cloning and sequence analysis of a bicistronic WDR53–CesA8A cDNA

Total RNA was extracted from 100 mg of xylem tissues and retro-transcribed as described above. PCRs were conducted to amplify bicis tronic cDNA fragments of different lengths as indicated using Gold Taq DNA polymerase (Applied Biosystems) and the primers listed in Supplementary Table S2 at JXB online. The amplified PCR products were cloned and sequenced. The sequence of the bicistronic WDR53–CesA8A cDNA was deposited in GenBank under the accession no. JQ272846.

Results and Discussion

Domesticated apple has 13 putative CesA genes

Mining of the recently sequenced apple genome (Velasco et al., 2010) showed the presence of 13 putative CesA genes, which code for proteins between 950 and 1330 amino acids long, displaying between six and nine predicted transmembrane helices (TMHs; Table 1). Taking into account the identities with the orthologues from A. thaliana and P. trichocarpa (64–91%), a nomenclature for the identified M. ×domestica CesA genes is proposed (Table 1). All of the identified genes also show the presence of the signature motif common to processive GT2s, which is predicted to be involved in substrate binding and catalysis (the four aspartic acid residues D, DxD, D, followed by the amino acids stretch QXXRW; Supplementary Fig. S1 at JXB online).

Table 1.

Proposed nomenclature (in bold and italics) for the CesA genes from M.×domestica (gene IDs are as reported in the Genome Database for Rosaceae GDR) based on amino acid identities with the orthologous proteins from A. thaliana and P. trichocarpa (identified by locus names)

M.×domestica Deduced polypeptide length TMHs A. thaliana % identity Arabidopsis–Malus P. trichocarpa % identity Populus–Malus
MdCesA1-A
MDP0000292881		 1283 9 CESA1 At4g32410 87 CESA1-A
POPTR_0018s01540		 91
MdCesA1-B
MDP0000279461		 1151 6 88 CESA1-B
POPTR_0006s26810		 91
MdCesA3-C
MDP0000314103		 1081 8 CESA3 At5g05170 85 CESA3-C
POPTR_0009s06560		 90
MdCesA3-D
MDP0000448752		 1071 8 86 CESA3-D
POPTR_0001s27320		 92
MdCesA4-A
MDP0000320351		 1054 8 CESA4 At5g44030 81 CESA4
POPTR_0002s25970		 88
MdCesA4-B
MDP0000313995		 1330 8 80 86
MdCesA6-B
MDP0000480237		 1097 8 CESA6 At5g64740 82 CESA6-B
POPTR_0007s07120		 88
MdCesA6-C
MDP0000792906		 1095 6 80 CESA6-C
POPTR_0005s21620		 81
MdCesA6-E
MDP0000291954		 1065 6 64 CESA6-E
POPTR_0013s02050		 77
MdCesA6-D
MDP0000185368		 1104 6 CESA9 At2g21770 79 CESA6-D
POPTR_0002s06710		 88
MdCesA8-A
MDP0000299896		 1307 8 CESA8 At4g18780 83 CESA8-A
POPTR_0011s07040		 84
MdCesA8-B
MDP0000214413		 981 6 82 CESA8-B
POPTR_0004s05830		 84
MdCesA7
MDP0000470441		 1077 8 CESA7 At5g17420 80 CESA7-B
POPTR_0018s11290		 82
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Phylogenetic and expression analyses of the 13 putative CesA genes from apple

Phylogenetic analysis of the CesA genes from M.×domestica, P. trichocarpa, and A. thaliana revealed the presence of six CESA clades in domesticated apple (Fig. 1), containing both primary (CESA1, 3, and 6; Desprez et al., 2007; Persson et al., 2007) and secondary cell wall biosynthesis classes (CESA4, 7, and 8; Taylor et al., 2003), in agreement with current models of CESA phylogeny (Holland et al., 2000; Nairn and Haselkorn, 2005). Figure 1 shows the phylogenetic tree obtained by aligning conserved amino acid regions within the second cytoplasmic loop, encompassing the U1–U4 regions, the QXXRW motif, and the HVR2 region, which was shown to be class selective (Carroll and Specht, 2011). Consistent with a recent (>50 million years ago) genome-wide duplication in apple (Velasco et al., 2010), a majority of the MdCesA genes appear as closely related gene pairs. Real-time PCR analysis carried out on different apple tissues largely confirmed the phylogenetic classification of M.×domestica CesA genes (Fig. 2): the genes belonging to the primary cell wall class (MdCesA1-A and B, MdCesA3-C, and MdCesA6-B, D, and E) showed the highest expression in buds (where meristematic tissue is present), while the genes involved in secondary cell wall biosynthesis (MdCesA4-A, MdCesA8-A and B, and MdCesA7) increased strongly in xylem. Within the category of primary cell wall CesA genes, the most abundantly expressed genes are MdCesA3-C, MdCesA6-D, and MdCesA1-A and B (note the log10 scale in Fig. 2A), whereas MdCesA6-B and E are expressed at low levels. Further, among the secondary cell wall CesA genes, MdCesA7, MdCesA8-B and MdCesA4-A show the highest expression levels (Fig. 2B), whereas MdCesA4-B and MdCesA8-A show low expression levels (Supplementary Discussion).

Fig. 1.

Fig. 1

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Phylogenetic relationships of CESAs from M.×domestica, P. trichocarpa, and A. thaliana by maximum likelihood analysis. Bootstrap=100. Numbers indicate branch support values. Mesotaenium caldariorum CESAI 1 (AAM83096) was used as the outgroup to root the tree. Secondary CESAs are framed in grey.

Fig. 2.

Fig. 2

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Real-time PCR expression analysis of MdCesA genes in different tissues. Charts show expression of (A) primary wall CesA genes, and of (B) secondary wall CesA genes. MNE, mean normalized expression.

A WD40 domain gene, WDR53, is a genomic neighbour of CesA8 in dicots

The bioinformatics analysis of the putative apple CesA genes revealed that the protein MdCESA8-A (MDP0000299896) is annotated as containing WD40 repeats at its N-terminus, an unlikely feature most probably reflecting incorrect annotation. By blasting the MdCESA8-A protein sequence against the plant gene family database at the Phytozome portal (http://www.phytozome.net), a similar WD40–CESA8 protein (XP_002522238) was identified in the genome of castor bean (Ricinus communis). The genomic sequences from both the M.×domestica and R. communis WD40–CesA8 genes were analysed, and it was found that in both species the boundary between the 10th exon and its immediate downstream intron is most probably wrongly annotated, as the intron DNA sequence codes for amino acid residues which are conserved in the C-termini of WD40 proteins, namely VYSVS* (R. communis) or VYSVA* (M.×domestica; Supplementary Fig. S2 at JXB online). It is therefore proposed that the WD40–CesA8 genomic regions in M.×domestica and R. communis encompass two tightly linked, co-oriented (→→) neighbouring genes, which code for two proteins, a WD40 protein and CESA8. The proteins encoded by the M.×domestica and R. communis CesA8-linked WD40 genes are members of a family of plant transducin/WD40 proteins, whose corresponding genes belong to Phytozome gene cluster 29002354 (represented by gene At5g45760 in Arabidopsis). This plant WD40 protein is highly similar to WDR53 (WD repeat protein 53), a mammalian protein of unknown function that was identified as an expressed gene in a retinoblastoma cDNA library (Strausberg et al., 2002). The plant WD40 protein is identified as WDR53 by SMART (http://smart.embl-heidelberg.de/) and therefore this protein is referred to as WDR53. It was observed that the WDR53–CesA8 collinear arrangement is strikingly conserved in dicot angiosperms, because 16 out of the 21 dicots in the Phytozome portal show this gene order and co-orientation (Fig. 3). Collinearity of WDR53 and CesA8 is an ancient feature in dicots, as it is present in the basal eudicot Aquilegia coerulea and in the basal dicot Mimulus guttatus. Phytozome dicot species that lack the WDR53–CesA8 collinear arrangement are the Brassicaceae species, Eucalyptus grandis, and Manihot esculenta.

Fig. 3.

Fig. 3

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Conserved microcollinearity of WDR53 and CesA8 in angiosperms. Species are indicated by their genus names. Species names are given in Supplementary Table S4 at JXB online. Unch, uncharacterized; EamA, EamA-like transporter; LRR leucine-rich repeat; PPR, pentatricopeptide repeat; Skp1, SCF ubiquitin ligase; RNUT1, snurportin 1.

WDR53 and CesA8 are partially co-expressed in the xylem

Based on the operon concept, a conserved genomic neighbourhood is considered an indicator of related protein function, mainly in prokaryotes, but also increasingly in eukaryotes (Gabaldón and Huynen, 2004). This also appears to be the case in plants, as adjacent neighbouring genes are frequently co-expressed in Arabidopsis (Williams and Bowles, 2004; Chen et al., 2010), and metabolic gene clusters have been detected in plants (Field et al., 2011). Then, the conserved collinearity of WDR53 and CesA8 in dicots could indicate that they are co-expressed and functionally related. CesA8 is xylem-specific and a member of the secondary cell wall gene program regulated by the transcription factors MYB46 and VND7 (Ko et al., 2009; Yamaguchi et al., 2011), but the expression of WDR53 is unknown. To establish whether WDR53 is to any extent co-expressed with CesA8, the expression profiles of WDR53 were investigated by quantitative real-time PCR on different apple tissues. The results showed that WDR53 displayed higher expression levels both in buds and in xylem (Fig. 4), suggesting that WDR53 and CesA8 are partially co-expressed in the apple xylem. Similar results were obtained by analysing microarray gene expression data in Populus and Arabidopsis (Supplementary Fig. S7 and Supplementary Discussion at JXB online).

Fig. 4.

Fig. 4

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Real-time PCR expression analysis of WDR53 in different tissues. The expression of MdCesA8-A, from Fig. 2, is added as a comparison. MNE, mean normalized expression.

A bicistronic WDR53–CesA8 mRNA in domestic apple contains a putative IRES element

In Arabidopsis, some adjacent and co-oriented genes are transcribed and spliced to generate bicistronic, or even fused monocistronic, RNAs (Thimmapuram et al., 2005; Xiao et al., 2005). Because the collinear arrangement of CesA8 and WDR53 is strikingly conserved in dicots, and the two genes are tightly linked (their genomic intergenic regions range between 700 bp and 3000 bp), fused WDR53–CesA8A mRNA was looked for in apple tissues by RT-PCR. Using xylem cDNA as template, it was possible to obtain a 730 bp PCR product encompassing the 3' end of WDR53 and the 5' end of CesA8A (Fig. 5A). Sequencing of this product revealed a 182 bp long transcribed IGS between the WDR53 and CesA8A cistrons, (Fig. 5A), and it was observed that a single fragment of 2022 bp, flanked by the canonical GT-AG splice sites, is spliced from the genomic IGS (Supplementary Fig. S3 at JXB online), indicating that the template giving rise to the 730 bp products is cDNA and not genomic DNA. However, the WDR53–CesA8A bicistronic mRNA is a rare transcript in the xylem, because the obtained PCR product was of low abundance. Because bicistronic transcripts may be accumulated in stressed tissues (Thimmapuram et al., 2005), cDNA from ‘Candidatus Phytoplasma mali’-infected apple trees was used as PCR template, and longer products encompassing the WDR53–CesA8A transcript were obtained (1026 bp and 1211 bp; shown in Fig. 5A and 5B). Using primers spanning over the translational start codon of WDR53 and the stop codon of CesA8A, a 3459 bp fragment was obtained that encompasses the complete WDR53 and CesA8A cistrons, and the sequence was deposited in GenBank (see the Materials and methods). The WDR53–CesA8A transcript is truly bicistronic, and not fused monocistronic, as it contains the WDR53 stop codon and therefore lacks an open reading frame encompassing both coding regions (Fig. 5C). Intriguingly, splicing of the 2022 bp fragment in the IGS removes the first exon and the first intron of MdCesA8-A, and the CESA8A-coding open reading frame starts at an alternative AUG start codon immediately upstream of the splicing site in the IGS (Fig. 5C; Supplementary Fig. S3), implying that the CesA8A cistron is alternatively spliced. As a consequence, the encoded protein, which was named altCESA8A, lacks a 59 amino acid long portion containing the zinc-binding domain (Supplementary Fig. S4). To better understand the expression of MdCesA8-A transcripts during biotic stress, real-time PCR was performed on tissues of phytoplasma-infected phloem, control (uninfected) phloem, and xylem, by using primers designed to discriminate between altCesA8A and MdCesA8-A (Fig. 6A, 6B). It is shown that, in the xylem, the most abundant transcript is MdCesA8-A, whereas altCesA8A is expressed at low levels. Further, in the infected phloem, altCesA8A is induced and is the dominant MdCesA8-A transcript. WDR53 is expressed in the three tissues (Fig. 6C), confirming its partial co-expression with MdCesA8-A in the xylem, and showing its partial co-expression with altCesA8A in the infected phloem. Further, WDR53 showed increased expression in infected apple phloem, in line with a role for this gene during stress conditions in domesticated apple. Finally, proteomic analysis of infected phloem tissues, involving excision of spots from two-dimensional polyacrylamide gels, trypsinization, and mass spectrometry analysis, failed to detect the presence of altCESA8A. This might be due to low abundance of the protein in infected tissues.

Fig. 5.

Fig. 5

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A bicistronic WDR53–CesA8A transcript in domestic apple. (A) Cartoon showing obtained WDR53–CesA8A RT-PCR fragments encompassing the intergenic spacer (IGS). RT-PCR was carried out using WDR53 forward and CesA8A reverse primers and tissues of xylem (black line) and phytoplasma-infected phloem (red lines). (B) Agarose gel showing the bicistronic RT-PCR products from phytoplasma-infected phloem as indicated in (A). (C) Sequence of the IGS between the WDR53 stop (UAG, in bold) codon and the altCESA8A start (AUG, in bold) codon. The site of intron splicing is indicated as opposing brackets. Alternative AUG sequences are underlined.

Fig. 6.

Fig. 6

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Real-time PCR expression analysis of MdCesA8-A (A), altCesA8A (B), and WDR53 (C) in control and phytoplasma-infected apple phloem, as well as xylem tissue. MNE, mean normalized expression.

It is shown that MdWDR53 and MdCesA8-A are collinear genes that display bicistronic expression in infected phloem of domestic apple. Because the IGS in the bicistronic transcript is 182 bp-long and contains two internal AUGs (shown in Fig. 5C), it is unlikely that ribosomes could translate the second cistron by continued scanning. However, altCesA8A translation could occur if the IGS contained an IRES. IRESs may be found in monocistronic and/or polycistronic viral and cellular mRNAs, and they may mediate cap-independent translation initiation in situations of cellular stress, when the cell’s standard cap-dependent translation initiation is compromised. Such cellular stresses include viral infection, mitosis, apoptosis, heat shock, hypoxia, and disease (Fitzgerald and Semler, 2009; Komar and Hatzoglou, 2011). IRES elements usually fold into highly structured RNA duplexes displaying multiple hairpins. To check whether an IRES could potentially be present between the WDR53 and the CesA8A cistrons in the spliced bicistronic transcript, the IGS was modelled using the M-fold program (Zuker, 2003), and hairpin structures that are characteristic of IRESs were obtained (Supplementary Fig. S5 at JXB online). The occurrence of an IRES within the IGS was further supported by analysis using the IRES search portal IRSS (http://140.135.61.9/ires/; Wu et al., 2009).

Phylogeny and predicted protein structure of WDR53

WDR53 is conserved in diverse eukaryotic groups, of which many, interestingly, produce cellulose. These latter include green plants, oomycetes, slime moulds, choanoflagellates, diatoms, and tunicates. In eukaryote genomes, WDR53 is present mostly as a one-copy gene, and its evolutionary relationships, inferred by phylogenetic analysis of WDR53 from different species (Fig. 7), with a few exceptions follow species phylogeny (Supplementary Discussion at JXB online). MdWDR53 is characterized by a relatively small size (371 amino acids long, theoretical molecular weight 40 681 Da), neutral pI, and six theoretical WD repeats as predicted by SMART, and these features are shared with plant and metazoan orthologues. Analysis of subcellular localization by WoLF PSORT (Horton et al., 2007) of seven dicot WDR53s predicted predominantly cytoplasmic localization. WD40 proteins may act as scaffolds during assembly of large protein complexes mediating multiple protein–protein interactions, and fold into a typical β-propeller structure, most commonly with seven blades. Domain analysis by SMART consistently detects six or fewer WD repeats in WDR53 proteins, but it has been proposed that one or more repeats often remain undetected by sequence-based classification methods (Stirnimann et al., 2010). The WDR53 3D structure was therefore modelled by homology modelling using the Phyre2 server (http://www.sbg.bio.ic.ac.uk/~phyre2/html; Kelley and Sternberg, 2009). The Phyre2 software identified WD40 proteins with a seven-bladed β-propeller structure as best templates for alignment (100% confidence), such as yeast β'-COP/COPB2, Arabidopsis RACK1, and yeast AIP1 (Voegtli et al., 2003; Ullah et al., 2008; Lee and Goldberg, 2010). From these models, it is inferred that WDR53 proteins fold into a seven-bladed β-propeller structure, where antiparallel β-strands, which form the blades of the propeller, are arranged around a central axis (Fig. 8A, 8B). Like other WD40 propellers, WDR53 forms a truncated cone, or frustum, as seen from the side (Fig. 8D). The frustum shape provides multiple surfaces for protein–protein interactions, including its top, bottom, and sides (Orlicky et al., 2003), and therefore plant WDR53s could potentially mediate protein–protein interactions in large protein complexes.

Fig. 7.

Fig. 7

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Phylogenetic relationships of WDR53 in eukaryotes by maximum likelihood analysis. Bootstrap=100. Numbers refer to percentage branch support values. Human RBBP4 (locus ENSP00000362592) was used as the outgroup to root the tree. Species are indicated by their genus names. Species name and gene/protein numbers are given in Supplementary Table S4 at JXB online.

Fig. 8.

Fig. 8

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Predicted 3D structure of plant WDR53 proteins based on the crystal structure of yeast COPB2 (Lee and Goldberg, 2010). (A) Homology model of M.×domestica WDR53, top view. (B) Homology model of P. trichocarpa XP_002316814, top view. (C) Crystal structure of the modelling template, the N-terminal β-propeller domain of COPB2 from yeast, top view. (D) The side view of the M.×domestica WDR53 protein model shows the frustum shape that characterizes WD40 propellers. All structures are shown as rainbow-coloured cartoons, where blue is the N-terminus and red is the C-terminus.

Potential functions of altCESA8A and WDR53

Taken together, the observations in this study suggest that alt-CESA8A, an alternatively spliced CESA8 protein lacking the zinc-binding domain, could be accumulated in infected phloem of domestic apple through cap-independent translation of a bicistronic WDR53–CesA8A transcript containing a putative IRES. IRES-containing cellular mRNAs are typically low abundant (Brogna et al., 1997), but they are more competitive for translation initiation when cap-dependent translation is suppressed, as only 3–5% of coding mRNAs remain associated with the polysomes under such conditions (Spriggs et al., 2008). Therefore, although the bicistronic transcript is not highly abundant in the infected phloem, its hairpin structure could function as an IRES, leading to competitive translation of altCESA8A in situations of cap-independent translation. Malus altCESA8A lacks the zinc-binding domain and therefore contains a shorter (114 amino acid long) cytoplasmic N-terminal domain consisting mainly of the hypervariable region I (HVRI; Delmer, 1999; Supplementary Fig. S6 at JXB online). Since this variant is found in phytoplasma-infected phloem, altCESA8A could be involved in stress responses. In the present study, alternative splicing of the bicistronic WDR53–CesA8A transcript leads to skipping of the first exon in the CesA8A cistron, removing one of the most intriguing domains in CESAs, the zinc-binding motif. The zinc motif was proposed to mediate dimerization of CESAs (Kurek et al., 2002), but interaction studies by a membrane-based yeast two-hybrid assay challenge this hypothesis (Timmers et al., 2009), and thus their function is still unclear. The altCESA8A protein could function during pathogen stress through either enhanced, or decreased, activity, as both scenarios could result in beneficial alterations of the cell wall. By way of an example, increased cellulose biosynthesis may result in reinforced cell walls with enhanced defence and/or mechanical properties. Conversely, decreased cellulose biosynthesis may induce defence mechanisms (Ellis et al., 2002; Caño Delgado et al., 2003; Hernandez Blanco et al., 2007). There are several possible mechanisms through which deletion of the zinc domain could either enhance or decrease cellulose biosynthesis. In the first scenario, where altCESA8A displays enhanced activity, it is possible that zinc motif deletion results in increased stability or increased catalytic activity, or both. Data showing that the zinc-binding domains of cotton fibre-expressed GhCesA1 display rapid turnover in vitro and in vivo (Jacob-Wilk et al., 2006) are consistent with increased stability of altCESA8A. However, these results could reflect rapid turnover of the zinc domain, rather than the whole enzyme, and zinc domain processing could be a mechanism for regulating CESA8-A activity, similar to the regulation of yeast Chs2 (chitin synthase 2), a transmembrane GT2 whose enzymatic activity increases upon proteolysis of the N-terminus (Martínez-Rucobo et al., 2009). Alternatively, zinc domain removal alters the spatial organization of the altCESA8A complexes, leading to reduced crystallinity and increased cellulose synthesis. Such a phenomenon was recently reported in a double CesA mutant of Arabidopsis, where mutations in the C-terminal transmembrane regions lead to reduced cellulose microfibril crystallinity and increased biosynthesis of cellulose (Harris et al., 2012), although the exact mechanism underlying this effect is unknown. In a second scenario, altCESA8A could function as a dominant-negative inhibitor of cellulose biosynthesis, by interfering with, or ‘poisoning’, the CESA complexes, as mutants of CesA7 exhibit dominant-negative effects in both Arabidopsis and rice (Zhong et al., 2003; Kotake et al., 2011).

Classification of WDR53 function is hampered by its lack of other domains besides WD repeats that could reveal its functional specificity, as can be found in other WD40 proteins that typically may contain F-box, protein kinase, or RING finger domains (van Nocker and Ludwig, 2003). The striking conservation of WDR53–CesA8-A collinearity in dicots hints at their functional relatedness, and it is tempting to speculate that WDR53 could have a role in cellulose biosynthesis. WDR53 expression overlaps with expression of CesA8A in the xylem, but its overall expression pattern is more extensive, suggesting that WDR53 may be multifunctional, a feature called moonlighting that is common in WD40 proteins. WD40 proteins that are similar to plant WDR53, by amino acid sequence or structurally, have roles such as scaffolding of proteins, microtubule interactions, turnover of proteins by ubiquitination, compartmentalization into lipid rafts at the plasma membrane, and trafficking and recycling of vesicles (examples summarized in Supplementary Table S3 at JXB online). Because cellulose synthesis involves precisely such mechanisms (reviewed in Guerriero et al., 2010), WDR53 could participate in this process by any of these roles (Supplementary Discussion).

Supplementary data

Supplementary data are available at JXB online.

Figure S1. Alignment of M.×domestica CesA genes.

Figure S2. Predicted exon–intron boundary at the 10th exon of two WD40–CesA8 gene models in M.×domestica and R. communis.

Figure S3. Genomic intergenic region of MdWDR53 and MdCesA8-A.

Figure S4. altCesA8A lacks the zinc-binding domain.

Figure S5. Predicted hairpin structure in the IGS of the WDR53–CesA8A bicistronic transcript

Figure S6. Predicted topology of altCESA8A.

Figure S7. Expression of WDR53 in A. thaliana and Populus tremula.

Table S1. Sequences of primers used in real-time PCR analysis.

Table S2. Sequences of nested primers used to amplify different fragments of the bicistronic WDR53–CesA8A transcript.

Table S3. Functions of WDR53-like WD40 proteins, and references.

Table S4. Names of species and corresponding WDR53 gene loci.

Supplementary Discussion.

Supplementary References.

Suppl. Mat.

Acknowledgements

The authors would like to thank Dr Silvia Schmidt for preparing the plant material used. Dr Christina Divne is gratefully acknowledged for advice on homology modelling. This study was funded by the Autonomous Province of Bozen/Bolzano, Italy (Departments 31 and 33). The authors acknowledge the South Tyrolean Fruit Growers’ Co-operatives, in particular VOG and VIP, for co-financing the Strategic Project on Apple Proliferation–APPL. GG was supported by the fellowship ‘Incoming researchers’ from the Autonomous Province of Bozen/Bolzano-South Tyrol (Promotion of Educational Policies, University and Research Department) and by the Austrian Science Fund (FWF): project number M1315. IE was supported by the Swedish Governmental Agency for Innovation Systems (VINNOVA).

References

  1. Arioli T, Peng L, Betzner AS, et al. Molecular analysis of cellulose biosynthesis in Arabidopsis. Science. 1998;279:717–720. doi: 10.1126/science.279.5351.717. [DOI] [PubMed] [Google Scholar]
  2. Baric S, Berger J, Cainelli C, Kerschbamer C, Letschka T, Dalla Via J. Seasonal colonisation of apple trees by ‘Candidatus Phytoplasma mali’ revealed by a new quantitative TaqMan real-time PCR approach. European Journal of Plant Pathology. 2011;129:455–467. [Google Scholar]
  3. Brogna S, Ashburner M. The Adh-related gene of Drosophila melanogaster is expressed as a functional dicistronic messenger RNA: multigenic transcription in higher organisms. EMBO Journal. 1997;16:2023–2031. doi: 10.1093/emboj/16.8.2023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caño-Delgado A, Penfield S, Smith C, Catley M, Bevan M. Reduced cellulose synthesis invokes lignification and defense responses in Arabidopsis thaliana. The Plant Journal. 2003;34:351–362. doi: 10.1046/j.1365-313x.2003.01729.x. [DOI] [PubMed] [Google Scholar]
  5. Carroll A, Specht CD. Understanding plant cellulose synthases through a comprehensive investigation of the cellulose synthase family sequences. Frontiers in Plant Genetics and Genomics. 2011;2:5. doi: 10.3389/fpls.2011.00005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen WH, de Meaux J, Lercher MJ. Co-expression of neighbouring genes in Arabidopsis: separating chromatin effects from direct interactions. BMC Genomics. 2010;11:178. doi: 10.1186/1471-2164-11-178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chevenet F, Brun C, Bañuls AL, Jacq B, Christen R. TreeDyn: towards dynamic graphics and annotations for analyses of trees. BMC Bioinformatics. 2006;7:439. doi: 10.1186/1471-2105-7-439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coutinho PM, Stam M, Blanc E, Henrissat B. Why are there so many carbohydrate-active enzyme-related genes in plants? Trends in Plant Science. 2003;8:63–65. doi: 10.1016/j.tplants.2003.10.002. [DOI] [PubMed] [Google Scholar]
  9. Delmer DP. Cellulose biosynthesis: exciting times for a difficult field of study. Annual Review of Plant Physiology and Plant Molecular Biology. 1999;50:245–276. doi: 10.1146/annurev.arplant.50.1.245. [DOI] [PubMed] [Google Scholar]
  10. Denness L, McKenna JF, Segonzac C, Wormit A, Madhou P, Bennett M, Mansfield J, Zipfel C, Hamann T. Cell wall damage-induced lignin biosynthesis is regulated by a reactive oxygen species- and jasmonic acid-dependent process in Arabidopsis. Plant Physiology. 2011;156:1364–1374. doi: 10.1104/pp.111.175737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Desprez T, Juraniec M, Crowell EF, Jouy H, Pochylova Z, Parcy F, Höfte H, Gonneau M, Vernhettes S. Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana. Proceedings of the National Academy of Sciences USA. 2007;104:15572–15577. doi: 10.1073/pnas.0706569104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Djerbi S, Lindskog M, Arvestad L, Sterky F, Teeri TT. The genome sequence of black cottonwood (Populus trichocarpa) reveals 18 conserved cellulose synthase (CesA) genes. Planta. 2005;221:739–746. doi: 10.1007/s00425-005-1498-4. [DOI] [PubMed] [Google Scholar]
  13. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research. 2004;32:1792–1797. doi: 10.1093/nar/gkh340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ellis C, Karafyllidis I, Wasternack C, Turner JG. The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses. The Plant Cell. 2002;14:1557–1566. doi: 10.1105/tpc.002022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Endler A, Persson S. Cellulose synthases and synthesis in Arabidopsis. Molecular Plant. 2011;4:199–211. doi: 10.1093/mp/ssq079. [DOI] [PubMed] [Google Scholar]
  16. Field B, Fiston-Lavier AS, Kemen A, Geisler K, Quesneville H, Osbourn AE. Formation of plant metabolic gene clusters within dynamic chromosomal regions. Proceedings of the National Academy of Sciences USA. 2011;108:16116–16121. doi: 10.1073/pnas.1109273108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fitzgerald KD, Semler BL. Bridging IRES elements in mRNAs to the eukaryotic translation apparatus. Biochimica et Biophysica Acta. 2009;1789:518–528. doi: 10.1016/j.bbagrm.2009.07.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fugelstad J, Bouzenzana J, Djerbi S, Guerriero G, Ezcurra I, Teeri TT, Arvestad L, Bulone V. Identification of the cellulose synthase genes from the Oomycete Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and enzyme activity. Fungal Genetics and Biology. 2009;46:759–767. doi: 10.1016/j.fgb.2009.07.001. [DOI] [PubMed] [Google Scholar]
  19. Gabaldón T, Huynen MA. Prediction of protein function and pathways in the genome era. Cellular and Molecular Life Sciences. 2004;61:930–944. doi: 10.1007/s00018-003-3387-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Grenville-Briggs LJ, Anderson VL, Fugelstad J, et al. Cellulose synthesis in Phytophthora infestans is required for normal appressorium formation and successful infection of potato. The Plant Cell. 2008;20:720–738. doi: 10.1105/tpc.107.052043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Guerriero G, Fugelstad J, Bulone V. What do we really know about cellulose biosynthesis in higher plants? Journal of Integrative Plant Biology. 2010;52:161–175. doi: 10.1111/j.1744-7909.2010.00935.x. [DOI] [PubMed] [Google Scholar]
  22. Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate large, phylogenies by maximum likelihood. Systematic Biology. 2003;52:696–704. doi: 10.1080/10635150390235520. [DOI] [PubMed] [Google Scholar]
  23. Hamann T, Denness L. Cell wall integrity maintenance in plants: lessons to be learned from yeast? Plant Signaling and Behavior. 2011;6:1706–1709. doi: 10.4161/psb.6.11.17782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Harris DM, Corbin K, Wang T, et al. Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase. Proceedings of the National Academy of Sciences USA. 2012;109:4098–4103. doi: 10.1073/pnas.1200352109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hématy K, Cherk C, Somerville S. Host–pathogen warfare at the plant cell wall. Current Opinion in Plant Biology. 2009;12:406–413. doi: 10.1016/j.pbi.2009.06.007. [DOI] [PubMed] [Google Scholar]
  26. Hernández-Blanco C, Feng DX, Hu J, et al. Impairment of cellulose synthases required for Arabidopsis secondary cell wall formation enhances disease resistance. The Plant Cell. 2007;19:890–903. doi: 10.1105/tpc.106.048058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Holland N, Holland D, Helentjaris T, Dhugga KS, Xoconostle-Cazares B, Delmer DP. A comparative analysis of the plant cellulose synthase (CesA) gene family. Plant Physiology. 2000;123:1313–1324. doi: 10.1104/pp.123.4.1313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, Nakai K. WoLF PSORT: protein localization predictor. Nucleic Acids Research. 2007;35:W585–W587. doi: 10.1093/nar/gkm259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jacob-Wilk D, Kurek I, Hogan P, Delmer DP. The cotton fiber Zn-binding domain of cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in vivo. Proceedings of the National Academy of Sciences USA. 2006;103:12191–12196. doi: 10.1073/pnas.0605098103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kelley LA, Sternberg MJ. Protein structure prediction on the Web: a case study using the Phyre server. Nature Protocols. 2009;4:363–371. doi: 10.1038/nprot.2009.2. [DOI] [PubMed] [Google Scholar]
  31. Ko JH, Kim WC, Han KH. Ectopic expression of MYB46 identifies transcriptional regulatory genes involved in secondary wall biosynthesis in Arabidopsis. The Plant Journal. 2009;60:649–665. doi: 10.1111/j.1365-313X.2009.03989.x. [DOI] [PubMed] [Google Scholar]
  32. Komar AA, Hatzoglou M. Cellular IRES-mediated translation: the war of ITAFs in pathophysiological states. Cell Cycle. 2011;10:229–240. doi: 10.4161/cc.10.2.14472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kotake T, Aohara T, Hirano K, Sato A, Kaneko Y, Tsumuraya Y, Takatsuji H, Kawasaki S. Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls. Journal of Experimental Botany. 2011;62:2053–2062. doi: 10.1093/jxb/erq395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kurek I, Kawagoe Y, Jacob-Wilk D, Doblin M, Delmer D. Dimerization of cotton fiber cellulose synthase catalytic subunits occurs via oxidation of the Zn-binding domains. Proceedings of the National Academy of Sciences USA. 2002;99:11109–11114. doi: 10.1073/pnas.162077099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lee C, Goldberg J. Structure of coatomer cage proteins and the relationship among COPI, COPII, and clathrin vesicle coats. Cell. 2010;142:123–132. doi: 10.1016/j.cell.2010.05.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Levin DE. Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews. 2005;69:262–291. doi: 10.1128/MMBR.69.2.262-291.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Martínez-Rucobo FW, Eckhardt-Strelau L, Terwisscha van Scheltinga AC. Yeast chitin synthase 2 activity is modulated by proteolysis and phosphorylation. Biochemical Journal. 2009;417:547–554. doi: 10.1042/BJ20081475. [DOI] [PubMed] [Google Scholar]
  38. Matthysse AG, Deschet K, Williams M, Marry M, White AR, Smith WC. A functional cellulose synthase from ascidian epidermis. Proceedings of the National Academy of Sciences, USA. 2004;101:986–991. doi: 10.1073/pnas.0303623101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Muller PY, Janovjak H, Miserez AR, Dobbie Z. Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques. 2002;32:1372–1374. 1378–1379. [PubMed] [Google Scholar]
  40. Nakashima K, Yamada L, Satou Y, Azuma J, Satoh N. The evolutionary origin of animal cellulose synthase. Development Genes and Evolution. 2004;214:81–88. doi: 10.1007/s00427-003-0379-8. [DOI] [PubMed] [Google Scholar]
  41. Nairn CJ, Haselkorn T. Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA genes from angiosperms. New Phytologist. 2005;166:907–915. doi: 10.1111/j.1469-8137.2005.01372.x. [DOI] [PubMed] [Google Scholar]
  42. Orlicky S, Tang X, Willems A, Tyers M, Sicheri F. Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase. Cell. 2003;112:243–256. doi: 10.1016/s0092-8674(03)00034-5. [DOI] [PubMed] [Google Scholar]
  43. Persson S, Paredez A, Carroll A, Palsdottir H, Doblin M, Poindexter P, Khitrov N, Auer M, Somerville CR. Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis. Proceedings of the National Academy of Sciences, USA. 2007;104:15566–15571. doi: 10.1073/pnas.0706592104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Richmond TA, Somerville CR. The cellulose synthase superfamily. Plant Physiology. 2000;124:495–498. doi: 10.1104/pp.124.2.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ringli C. Monitoring the outside: cell wall-sensing mechanisms. Plant Physiology. 2010;153:1445–1452. doi: 10.1104/pp.110.154518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sanderson K. Lignocellulose: a chewy problem. Nature. 2011;474:S12–S14. doi: 10.1038/474S012a. [DOI] [PubMed] [Google Scholar]
  47. Saxena IM, Kudlicka K, Okuda K, Brown RM., Jr Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization. Journal of Bacteriology. 1994;176:5735–5752. doi: 10.1128/jb.176.18.5735-5752.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sieber P, Schorderet M, Ryser U, Buchala A, Kolattukudy P, Métraux JP, Nawrath C. Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions. The Plant Cell. 2000;12:721–738. doi: 10.1105/tpc.12.5.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Spriggs KA, Stoneley M, Bushell M, Willis AE. Re-programming of translation following cell stress allows IRES-mediated translation to predominate. Biology of the Cell. 2008;100:27–38. doi: 10.1042/BC20070098. [DOI] [PubMed] [Google Scholar]
  50. Steinwand BJ, Kieber JJ. The role of receptor-like kinases in regulating cell wall function. Plant Physiology. 2010;153:479–484. doi: 10.1104/pp.110.155887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Stirnimann CU, Petsalaki E, Russell RB, Müller CW. WD40 proteins propel cellular networks. Trends in Biochemical Sciences. 2010;35:565–574. doi: 10.1016/j.tibs.2010.04.003. [DOI] [PubMed] [Google Scholar]
  52. Strausberg RL, Feingold EA, Grouse LH, et al. Generation and initial analysis of more than 15,000, full-length human and mouse cDNA sequences. Proceedings of the National Academy of Sciences, USA. 2002;99:16899–16903. doi: 10.1073/pnas.242603899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Svagan AJ, Azizi Samir MAS, Berglund LA. Biomimetic polysaccharide nanocomposites of high cellulose content and high toughness. Biomacromolecules. 2007;8:2556–2563. doi: 10.1021/bm0703160. [DOI] [PubMed] [Google Scholar]
  54. Taylor NG, Howells RM, Huttly AK, Vickers K, Turner SR. Interactions among three distinct CesA proteins essential for cellulose synthesis. Proceedings of the National Academy of Sciences, USA. 2003;100:1450–1455. doi: 10.1073/pnas.0337628100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Thimmapuram J, Duan H, Liu L, Schuler MA. Bicistronic and fused monocistronic transcripts are derived from adjacent loci in the Arabidopsis genome. RNA. 2005;11:128–138. doi: 10.1261/rna.7114505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Timmers J, Vernhettes S, Desprez T, Vincken JP, Visser RG, Trindade LM. Interactions between membrane-bound cellulose synthases involved in the synthesis of the secondary cell wall. FEBS Letters. 2009;583:978–982. doi: 10.1016/j.febslet.2009.02.035. [DOI] [PubMed] [Google Scholar]
  57. Ullah H, Scappini EL, Moon AF, Williams LV, Armstrong DL, Pedersen LC. Structure of a signal transduction regulator, RACK1, from Arabidopsis thaliana. Protein Science. 2008;17:1771–1780. doi: 10.1110/ps.035121.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. van Nocker S, Ludwig P. The WD-repeat protein superfamily in Arabidopsis: conservation and divergence in structure and function. BMC Genomics. 2003;4:50. doi: 10.1186/1471-2164-4-50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Velasco R, Zharkikh A, Affourtit J, et al. The genome of the domesticated apple (Malus × domestica Borkh.) Nature Genetics. 2010;42:833–839. doi: 10.1038/ng.654. [DOI] [PubMed] [Google Scholar]
  60. Voegtli WC, Madrona AY, Wilson DK. The structure of Aip1p, a WD repeat protein that regulates Cofilin-mediated actin depolymerization. Journal of Biological Chemistry. 2003;278:34373–34379. doi: 10.1074/jbc.M302773200. [DOI] [PubMed] [Google Scholar]
  61. Williams EJ, Bowles DJ. Coexpression of neighboring genes in the genome of Arabidopsis thaliana. Genome Research. 2004;14:1060–1067. doi: 10.1101/gr.2131104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wu TY, Hsieh CC, Hong JJ, Chen CY, Tsai YS. IRSS: a web-based tool for automatic layout and analysis of IRES secondary structure prediction and searching system in silico. BMC Bioinformatics. 2009;10:160. doi: 10.1186/1471-2105-10-160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Xiao YL, Smith SR, Ishmael N, Redman JC, Kumar N, Monaghan EL, Ayele M, Haas BJ, Wu HC, Town CD. Analysis of the cDNAs of hypothetical genes on Arabidopsis chromosome 2 reveals numerous transcript variants. Plant Physiology. 2005;139:1323–1337. doi: 10.1104/pp.105.063479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yamaguchi M, Goué N, Igarashi H, et al. VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 effectively induce transdifferentiation into xylem vessel elements under control of an induction system. Plant Physiology. 2010;153:906–914. doi: 10.1104/pp.110.154013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zhang Q, Pettolino FA, Dhugga KS, et al. Cell wall modifications in maize pulvini in response to gravitational stress. Plant Physiology. 2011;156:2155–2171. doi: 10.1104/pp.111.179606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zhong R, Morrison WH, 3rd, Freshour GD, Hahn MG, Ye ZH. Expression of a mutant form of cellulose synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis. Plant Physiology. 2003;132:786–795. doi: 10.1104/pp.102.019331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research. 2003;31:3406–3415. doi: 10.1093/nar/gkg595. [DOI] [PMC free article] [PubMed] [Google Scholar]

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