Molecular cloning and expression analysis of multiple polyphenol oxidase genes in developing wheat (Triticum aestivum) kernels

Journal of Cereal Science 53 (2011) 371e378

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Molecular cloning and expression analysis of multiple polyphenol oxidase genes in developing wheat (Triticum aestivum) kernelsq Brian Beecher*, Daniel Z. Skinner USDA-ARS, Wheat Genetics, Quality, Physiology and Disease Research Pullman, WA 99164-6394, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 September 2010 Received in revised form 16 December 2010 Accepted 6 January 2011

Polyphenol oxidase (PPO) is a major cause of time-dependent discoloration in raw wheat (Triticum aestivum) flour dough. The PPO-A1 and PPO-D1 genes have previously been implicated in dough discoloration. However, wheat contains multiple PPO genes. The goal of this study was to identify and quantify expression levels for PPO genes relevant to wheat quality. Three novel sequences were identified and found to be orthologous to one another and paralogous to the previously described PPO-A1/PPO-D1 group. The new genes localized to homeologous group 2 chromosomes. We propose naming these new genes PPO-A2, PPO-B2, and PPO-D2. Real-time PCR analysis determined that in the wheat cultivar ‘Alpowa’, PPO-A1a, PPO-A2b, PPO-D1b and PPO-D2b were all expressed to substantial levels in developing wheat kernels, while PPO-B2b was not. Transcript levels varied over the course of grain development, with peak levels observed at 9e16 days post-anthesis. These results show that wheat kernel PPO activity is the result of at least two orthologous families of two paralogous genes and that some of these genes are expressed to several-fold greater levels than others. The novel PPO-2 genes described here together account for 72% of PPO transcripts in developing kernels of the wheat cultivar Alpowa. Published by Elsevier Ltd.

Keywords: Triticum aestivum Noodle discoloration Polyphenol oxidase (PPO) Chromosomal location

1. Introduction Polyphenol oxidase (PPO) is a ubiquitous copper-containing enzyme causing browning reactions in many plant-based products, including those made from cereal grains (Whitaker and Lee, 1995). PPO activity is the largest contributor to time-dependent discoloration in wheat based products, particularly yellow alkaline and white-salted noodles (Baik et al., 1994; Fuerst et al., 2010). PPOs catalyze the hydroxylation and dehydrogenation of phenolic compounds to o-quinones(EC 1.14.18.1, EC 1.10.3.1); o-quinone products react with amines and thiol groups or undergo self polymerization to form melanins, responsible for food product darkening (Mayer and Harel, 1979). Products with a larger degree of discoloration are considered less desirable to consumers (Mares and Panozzo, 1999; Morris et al., 2000). Wheat improvement

Abbreviations: PPO, polyphenol oxidase; EST, expressed sequence tag; dpa, days post-anthesis; T. aestivum, Triticum aestivum L. subsp. aestivum. q Mention of trademark or proprietary products does not constitute a guarantee or warranty of a product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. This article is in the U.S. Public Domain and is not copyrightable. * Corresponding author. Tel.: þ509 335 4062; fax: þ509 335 8573. E-mail address: [email protected] (B. Beecher). 0733-5210/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.jcs.2011.01.015

efforts seek to minimize grain PPO activity levels to produce a more marketable product. This has proven difficult because bread wheat is an allohexaploid composed of three homeologous genomes, which provide genetic redundancy. In addition, wheat has been found to contain multiple paralogous PPO genes, similar to that of several other plant species (Gooding et al., 2001; Newmann et al., 1993; Sullivan et al., 2004; Thygesen et al., 1995). Paralogs are genes that occur within a genome and are descended from a gene duplication event. Orthologs, in contrast are found in different genomes and have evolved from a common ancestral gene through speciation. The existence of paralogous PPO loci in wheat was first proposed by Jukanti et al. (2004), who found that six PPO ESTs could be arranged in two distinct phylogenetic groups containing three members each. The groups could be further differentiated by expression type based on the tissue from which they were isolated (Jukanti et al., 2004). Members of one group are not known to be expressed in developing seeds (i.e. the “non-kernel” cluster), while the other group was isolated from developing seed libraries (the “kernel-type” cluster) (Anderson et al., 2006; Jukanti et al., 2004). These data were consistent with two PPO genes arising in the ancestral diploid progenitor from a gene duplication event (Jukanti et al., 2004). These two paralogs would then have evolved into three orthologous members each (one for each diploid genome of hexaploid wheat), for a total of six PPO genes. Massa et al. (2007)

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cloned and sequenced 21 distinct partial PPO sequences spanning a variable region of the genes. These sequences formed four phylogenetic groups within the ‘kernel-specific’ cluster, indicating that multiple paralogous PPO genes likely exist in hexaploid wheat (Massa et al., 2007). One of these ‘kernel-specific’ paralog families, represented by the PPO-A1 and PPO-D1 genes, has been well described (Chang et al., 2007; He et al., 2007, 2009; Sun et al., 2005). PPO-A1 and PPO-D1 are located on the long arm of the homeologous chromosomes 2A and 2D, respectively (Anderson et al., 2006; Chang et al., 2007; Demeke et al., 2001; He et al., 2007; Jimenez and Dubcovsky, 1999; Mares and Campbell, 2001; Raman et al., 2005; Simeone et al., 2002; Sun et al., 2005; Watanabe et al., 2004; Zeven, 1972; Zhang et al., 2005). These findings are in agreement with previous work that localized kernel PPO activity to chromosomes 2A and 2D (Anderson et al., 2006; Chang et al., 2007; Demeke et al., 2001; He et al., 2007; Jimenez and Dubcovsky, 1999; Mares and Campbell, 2001; Raman et al., 2005, 2007; Simeone et al., 2002; Sun et al., 2005; Watanabe et al., 2004; Wrigley and McIntosh, 1975; Zeven, 1972; Zhang et al., 2005). Allelic variation at PPO-A1 and PPO-D1 is associated with kernel PPO activity (Chang et al., 2007; He et al., 2007; Sun et al., 2005). However, work by Massa et al. (2007) demonstrated that multiple genes closely related to PPO-A1 and PPO-D1 exist within the wheat genome. The goals of this study were four fold: (1) unequivocally demonstrate that more than two paralogous PPO loci exist in hexaploid wheat by isolating multiple full-length clones from single cultivars; (2) characterize these clones’ gene structure and phylogenetic relationships, (3) determine their chromosomal location(s); and (4) quantify their expression levels in developing wheat kernels. 2. Experimental 2.1. Plant material Cytogenetic stocks of the wheat cultivar ‘Chinese Spring’ (CS) developed by Sears (1966) were kindly provided by Mr. John Raupp, Wheat Genetics and Genomics Research Center, Kansas State University, USA. In addition, the Triticum aestivum wheat cultivars Alpowa, ‘UC1110’, ‘CIMMYT-2, ‘Rio Blanco’, ‘IDO444’, ‘IDO556’, ‘Zak’, ‘Louise’, ‘Penewawa’, ‘Weebil 1’, ‘Jupateco 73S’, ‘SS550’, ‘Pioneer Variety 26R46’, ‘P91193’, ‘P92201’, ‘Cayuga’, ‘Caledonia’, ‘Platte’, ‘CO940610’, ‘Tam 105,’ and ‘Jagger’ were included in the study. Wheat plants were grown in pots in a greenhouse at 20e25  C under 16/8 h day/night photoperiod with supplemental lighting. Cultivation for DNA isolation proceeded until the 2 leaf stage, at which time the tissue was harvested. The spring wheat cultivar Alpowa was chosen for expression analysis. Seeds were individually marked at anthesis for later collection in the time course experiment. Tissue from duplicate plants was harvested and immediately frozen under liquid nitrogen and stored at 80  C until RNA isolation. 2.2. Nucleic acid isolation and cDNA preparation Genomic DNA was isolated by the method of Riede and Anderson (1996). Total RNA was isolated from flash-frozen tissue by grinding under liquid nitrogen in a mortar and pestle. RNA extraction was conducted using ‘Tri-Reagent’Ò (Molecular Research Center, Inc) using the company’s protocol. The integrity and quantity of RNA were visually estimated compared to known standards using 1% Agarose gels in 1 TAE buffer. In addition, the total RNA isolated from developing seeds for real-time PCR analysis was treated with an additional Deoxyribonuclease I (DNaseI; Promega, Madison, WI) step to remove contaminating genomic

DNA prior to reverse transcription. cDNA was prepared from 2 mg of total RNA using the Verso cDNA kit (Thermo Scientific, Waltham, MA) with the following important modification: after the initial 25 min incubation at 42 C, Betaine was added to a final concentration of 1 M and the reaction allowed to proceed for an additional 20 min at 50  C. 2.3. Polymerase chain reaction and DNA sequencing Allele-specific PCR was conducted with the following protocol: The primers described in Table 1 were used to amplify products from approximately 50 ng of a genomic or cDNA preparation using Taq DNA polymerase (5 Prime, Gaithersburg, MD) (Riede and Anderson, 1996). The temperature regimen consisted of a 3 min initial denaturation step at 95  C, followed by forty cycles of 95  C for 50 s, 30 s at the annealing temperature (see Table 1), and a 90 s extension step at 72  C. Reaction conditions consisted of 1 PCR buffer (New England Biolabs), 0.2 mM each dNTP, 0.2 pmol/mL of each primer, 1 M Betaine, and 0.02 U/ml Taq polymerase in a 25 ml reaction volume using a PTC-200 Thermo Cycler (MJ Research). The genomic clones reported here were amplified from T. aestivum cultivars Chinese Spring and Alpowa genomic DNA using the primers POUT5S4and POUT53A1 (Supplementary Table 1) via the proofreading PhusionÒ polymerase (Espoo, Finland). Phusion cycling conditions consisted of a 30 s initial denaturation step at 98  C, followed by forty cycles of 98  C for 10 s, annealing at 50  C for 30 s, with a 2-min extension step at 72  C. Reaction conditions consisted of 1 Phusion GC PCR buffer, 0.2 mM each dNTP, 0.2 pmol/ml of each primer, 3% (v/v) DMSO, and 0.01 U/ml Phusion polymerase in a 25 ml reaction volume using an MJ Research PTC200 Thermo Cycler. The proofreading PCR products were purified using AxyPrep gel extraction kit columns (Axygen Scientific, Union City, CA) brought to 1 Taq cycling conditions, and incubated at 72  C for 10 min to add single adenosine extensions. These ‘A-tailed’ PCR products were ligated to pGEMT-Easy (Promega, Madison, WI). Approximately 92 individual clones for each cultivar were screened by sequencing. The identity of each reported sequence was verified by re-sequencing from an independent PCR clone. The purified PCR products were sequenced using the BigDyeÒ Terminator Cycle Sequencing Ready Reaction Kit (PerkineElmer). The sequencing reactions were precipitated with ethanol and EDTA and run on an Applied Biosystems 3130 Genetic Analyzer. Sequence reads were analyzed using the programs Finch TV and Biology Workbench 3.2 (http://workbench.sdsc.edu). 2.4. Real-time PCR The concentrations of the cDNA products were measured as single-strand DNA using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The PCRs were

Table 1 Sequence comparison of six haplotypes of the PPO-2 genes. DNA sequence comparisons are shown below the diagonal, while inferred amino acid sequence comparisons are shown above. Relationships are shown as % identity. Allele

PPO-A2a PPO-A2b PPO-B2a PPO-B2b PPO-D2a PPO-D2b [clone 7] [clone 1] [clone 4] [clone 3] [clone 5] [clone 2]

PPO-A2a [clone 7] PPO-A2b [clone 1] PPO-B2a [clone 4] PPO-B2b [clone 3] PPO-D2a [clone 5] PPO-D2b [clone 2]

* 99.9 88.7 88.8 95.1 89.0

100 * 88.7 88.8 95.1 89.0

94.5 94.5 * 99.7 89.3 96.2

95.0 95.0 99.5 * 89.3 96.4

97.5 97.5 94.3 94.8 * 88.7

95.0 95.0 96.1 96.6 95.2 *

The Six haplotypes PPO-A2a, PPO-A2b, PPO-B2a, PPO-B2b, PPO-D2a, PPO-D2b, have been deposited in GenBank, accessions # through #, respectively.

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conducted on an Applied Biosystems (Foster City, CA USA) 7300 Real-Time PCR System using 25-ml preparations that consisted of 1 Go-TaqÒ Colorless Master Mix (Promega, Madison, WI, USA), 1 ml cDNA, 200 mM of each primer, 0.85 SYBR Green I (Invitrogen, Carlsbad, CA, USA), and 300 nM ROX dye (Roche, Indianapolis, IN, USA) as a passive fluorescence standard. The qPCR software available with the instrument was used to normalize the fluorescence data and to determine the Ct, the fractional cycle number at which the fluorescence intensity reached an arbitrary threshold, using the threshold determined by the software. The PCR primers used for each gene are shown in Supplementary Table 1. Replicons were 100 base pairs in length for each gene. Two independent qPCR replications of each of two independent biological samples collected at each time point were performed. The PCR temperature profile used was an initial dissociation step at 95  C for 2 min, then 50 cycles of 95  C for 15 s, followed by a primer annealing/primer extension step at 60  C for 45 s. At the end of the 50 amplification cycles, the dissociation curves of the PCR products were generated and examined to confirm that a single PCR product had been amplified. Transcript copy numbers were estimated using the absolute quantification method described by Pfaffl and Hageleit (2001). The amplicon resulting from the PPO-A2b primers shown in Supplementary Table 1 was cloned into pGEMT-Easy (Promega, Madison, WI) and used as template to generate the calibration curve. The number of copies of DNA molecules used to develop the curve ranged from an estimated 3.1 copies to 3.1  108 copies in a 10-fold, serial dilution series. This calibration curve process was repeated as two independent replications. A linear regression equation relating Ct to the logarithm of the initial number of copies was found (R2 ¼ 0.982; not shown). This equation was used to estimate the number of copies of each target PPO gene in the cDNA samples. The copy numbers were then expressed on a per ng cDNA basis. 2.5. Phylogenetic analysis All sequences were aligned with Clustal X, version 2.0.12 (Larkin et al., 2007). Gene trees were generated by maximum likelihood and neighbor-joining algorithms. Bootstrap was performed using 1000 replicates. Phylogenetic trees were constructed using TreeView (Page, 1996). Intron splice sites were inferred based on sequence comparison with sequenced cDNA clones of PPO-A2a and PPO-D2a from T. aestivum cv Chinese Spring and the GenBank accession BT009357. 3. Results 3.1. Identification of five distinct kernel-type PPO sequences in individual T. aestivum cultivars by gene cloning Previous work indicated the possibility that multiple paralogous genes might exist within the kernel sequence type (Massa et al., 2007). The primers POUT5S4and POUT53A1 (Supplementary Table 1) were designed to specifically amplify the gene represented by GenBank accession BT009357; however, direct sequencing of the PCR product revealed a mixture of distinct sequences. These observed polymorphisms were further examined by subsequent sequencing of individual clones. Six distinct and novel sequences were found; three occurred in each cultivar. These are represented in the sequence drawings of Fig. 1. All contain a complete open reading frame encoding a polypeptide similar to previously reported polyphenol oxidase sequences (Bucheli et al., 1996; Cary et al., 1992; Gooding et al., 2001; Hunt et al., 1993). Two catalytic copper-binding sites as well as the thylakoid targeting sequence are present and well conserved relative to known PPO genes (Supplementary Fig. 1) (Cary et al., 1992; Sommer et al., 1994;

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Van Gelder et al., 1997). Chinese Spring clone 7 and Alpowa clone 1 contain a single intron of 222 bp and have first and second exon lengths of 557 bp and 1117 bp respectively. Chinese Spring clone 5 is identical in structure to the above with the exception that its intron is 213 bp in length. The open reading frames of these three sequences are all 1671 bp in length and encode 557-residue polypeptides (Fig. 1). Chinese Spring clone 4 and Alpowa clone 3 are identical in structure and contain two introns. Exons 1, 2, and 3 are 572, 262, and 855 bp in length, introns I and II comprise 202 and 98 bp, respectively. The structure of Alpowa clone 2 is identical to that of the previous two clones, with the exception that intron I is 203 bp in length. Chinese Spring clone 4, and Alpowa clones 2 and 3 all have 1686 bp open reading frames and encode 562 amino acid polypeptides. The coding region length differences among these clones is due to a single 15 bp insertion contained in the 50 region of exon 1 in Alpowa clones 2 and 3, and Chinese Spring clone 4 (Fig. 1). A total of eight insertions and deletions were noted between these novel PPO clones and that of the previously described PPO-A1b and PPO-D1a. These indels were located on the distal regions, away from the catalytic central portion of the encoded enzyme (Fig. 1). In addition, the primer sets (PPOA1aNtF6/PPOA1ainR7) and (PPOAlainF6/PPOA1aCtR5a) were used to confirm the presence and sequence of PPO-A1a and PPO-A1b in Alpowa and Chinese Spring, respectively (Supplementary Table 1). The primer sets (PPOD1aNtF1/PPOD1aint2R1) and (PPOD1aint2F1/PPOD1aCter2R1) were used to confirm the presence and sequence of PPO-D1a in Chinese Spring (Supplementary Table 1). The primer sets (PPOD1bNtF6/PPOD1binR1a) and (PPOD1binF1a/PPOD1bCtR1a) were used to confirm the presence and sequence of PPO-D1b in Alpowa (Supplementary Table 1). It was confirmed that as reported by He et al., (2007), ‘Chinese Spring contained the PPO-A1b and PPO-D1a sequences. Alpowa was found to contain the PPO-A1a and PPO-D1b alleles (data not shown). The complete sequences of the six novel clones reported here were deposited in GenBank as follows: PPO-A2a(aka clone 7), HQ228148; PPO-A2b (aka clone 1), HQ228149; PPO-B2a(aka clone 4), HQ228150; PPO-B2b(aka clone 3), HQ228151; PPO-D2a(aka clone 5), HQ228152; PPO-D2b(aka clone 2), HQ228153. 3.2. Evolutionary relationships among Wheat PPO sequences Phylogenetic analysis was conducted on a dataset consisting of sixteen PPO sequences excluding introns and gaps. This set included the six newly-described clones from Chinese Spring and Alpowa, as well as ten accessions from GenBank. These accessions included the well characterized PPO-A1a, PPO-A1b, PPO-D1a, and PPO-D1b genes (EF070147, EF070148, EF070149, and EF070150, respectively). The uncharacterized ‘kernel-type’ sequences BT009357, and AY596269 as well as a non-kernel-type sequence (AF507945) were included in the analyses. Due to the partial nature of some of the comparative sequences, the regions polymorphic between the PPO-A2a, PPO-A2b, and BT009357 were not included in constructing the tree. The same holds true for the group comprising PPO-D1a, AY515506, and AY596270. Three major clusters can be inferred, one of which contains highly divergent, non-kernel-type PPO sequence class (Fig. 2A), while two correspond to the kerneltype PPO class (Fig. 2B and C). The major clusters themselves can be further subdivided (bootstrap values of 100%). Cluster B contains the previously described PPO genes as well as the accessions AY515506 and AY596270. The alleles at the PPO-A1 locus are more similar than those of the PPO-D1 locus. The genetic distances between PPO-D1a and PPO-D1b are nearly as great as the distance from either of these genes to PPO-A1 (Fig. 2B), making them appear as orthologous sequences. However, previous work has demonstrated that PPO-D1a and PPO-D1b behave as haplotypes (He et al.,

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Fig. 1. Structures of cloned members of the new paralogous gene family from Triticum aestivum. (A). Newly isolated PPO sequences isolated from T. aestivum cv. Chinese Spring and Alpowa (this work). (B). Previously characterized PPO sequences from T. aestivum cv. Chinese Spring PPO-A1b (EF070148) and PPO-D1b (EF070149) (He et al., 2007).

2007). Cluster C (Fig. 2C) contains the newly-described clones, as well as the uncharacterized GenBank accessions BT009357 and AY596269. Five well-separated groups (bootstrap values >80%) were observed. Alpowa clone 1, Chinese Spring clone 7, and accession BT009357 are very similar, and are potentially allelic sequences. Likewise, Alpowa clone 3 and Chinese Spring clone 4 are very similar to one another. The remaining three groups have a single representative each, namely Alpowa clone 2, Chinese Spring clone 5, and AY596269. Genetic distances among the groups of cluster C are similar to but slightly less than those of Cluster B (Fig. 2B and C). Overall the relationships among the members of cluster B and cluster C are similar in that there are a few groups of closely-related potentially allelic sequences that are distributed among more distantly related sequences within the group.

3.3. Chromosomal locations of the new paralogous group members The results shown in Fig. 3 clearly indicate the chromosomal locations of Chinese Spring clones 4, 5, and 7. The PPOCln7F1/ PPOCln7R1 primer set (Supplementary Table 1) amplified the expected 404 bp product from all samples with the exception of lane 4, which contained the DT2AS genomic sample (Fig. 3A, first gel). Therefore we can conclude that the sequence corresponding to Chinese Spring Clone 7 is located on the long arm of Chromosome 2A. The PPOCln4F1/PPOCln4R1 primer set (Supplementary Table 1) amplified its 493 bp product from all samples except lane 5, which contained N2B-T2A (Fig. 3A second gel). Clone 4 therefore resides on chromosome 2B. Since the N2B-T2A sample is a complete

chromosomal substitution, clone 4 cannot be localized to a specific chromosome arm. The PPOCln5F1/PPOClns2n5R1 primer set (Supplementary Table 1) amplified its expected 275 bp clone 2 product from all lanes except lane 6, which contained the DT2DS genomic sample (Fig. 3A third gel). We can therefore conclude that the sequence corresponding to Chinese Spring Clone 5 is located on the long arm of Chromosome 2D. Based on these chromosomal locations, the newly-described PPO genes were renamed as follows: Chinese Spring clone 7 is now PPO-A2a, Chinese Spring clone 4 is now PPO-B2a, and Chinese Spring clone 5 is now PPO-D2a.

3.4. Allelic assignment of AlpowaPPO-2 sequences Sequence comparisons clearly indicate the likely allelic assignment for the PPO-A2 and PPO-B2 alleles from Alpowa. Clone 1 from Alpowa is 99.9% identical to PPO-A2 (clone 7) at the nucleic acid level, and due to silent mutations, encodes an identical polypeptide (Table 1). Therefore we rename Alpowa clone 1 as PPO-A2b. Alpowa clone 3 shows 99.7% identity at the nucleic acid level with that of PPO-B2a (clone 4). This equates to 99.5% at the amino acid level (Table 1). Alpowa Clone 3 is therefore determined to be allelic to PPO-B2a, and is now renamed PPO-B2b. However, clone 2 from Alpowa cannot be assigned as an allele of PPO-D2 on the basis of sequence similarity to PPO-D2a. Clone 2 shows 89.0%, 96.2%, and 88.7% nucleic acid identity to PPO-A2a, PPO-B2a, and PPO-D2a, respectively. It thus shows more similarity to PPO-B2a than to PPOD2a. Fig. 1 shows that clone 2 contains the 50 insertion as well as the second intron, both of which are present in PPO-B2 (Alpowa clones

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Fig. 2. Phylogenetic analysis of the newly identified PPO sequences. The tree was derived by neighbor-joining distance analysis using the novel PPO sequences isolated from the cultivars Chinese Spring and Alpowa as well as the previously described genes PPO-A1a, PPO-A1b, PPO-D1a, and PPO-D1b. The GenBank accessions AF507945, BT009357, AY515506, AY596269, and AY596270 were also included in the analysis. Bootstrap values over 70% are indicated. The non-kernel-specific sub-tree (A) was regrafted to a divergent scale and appended to the main tree as indicated by a broken horizontal line. The branch of sub-tree A and it’s scale bar are shown by a dotted line. (B) and (C) show the two branches of kernel-specific PPO sequences. Sequences are labeled by GenBank accessionnumber and gene name (in parentheses). Scale bars indicate the number of nucleotide substitutions per site.

3 and 4) and are absent in PPO-A2 and PPO-D2a (Alpowa clones 7, 1, and 5, respectively). Comparison at the amino acid level showed that clone 2 is roughly similar to the other PPO-2 genes, (95.0%, 96.1%, and 95.2% identical to PPO-A2a, PPO-B2a and PPO-D2a respectively, Table 1). Although clearly a member of this paralogous group (Figs. 1 and 2), clone 2 has the appearance of being an orthologous gene to PPO-A2, PPO-B2, and PPO-D2. To determine the relationship between PPO-D2a and clone 2, primer sets specific to PPO-D2a (PPOCln5F1/PPOCln5R1) and Alpowa clone 2 (PPOCln2F1/ PPOClns2n5R1) were designed and used to analyze twenty-two randomly chosen wheat cultivars by sequence-specific PCR (Fig. 3B). In every case, either the clone 2 sequence or the PPO-D2a sequence was amplified. In no case were both sequences detected in the same genotype (Fig. 3B). This analysis was expanded to include a total of 124 genotypes. Seventy-three genotypes, including the cultivars CIMMYT-2, Rio Blanco, IDO444, IDO556, Zak, Louise, Weebil 1, SS550, Pioneer Variety 26R46, Platte, CO940610, and Tam 105 contained PPO-D2a specific PCR product, while 51 genotypes, including the cultivars Alpowa, UC1110, Penewawa, Jupateco 73S, P91193, P92201, Cayuga, Caledonia, Jagger contained PPO-D2b specific PCR product. In no instance were PPO-D2a and PPO-D2b both present in a single cultivar (data not shown). We therefore conclude that clone 2 and PPO-D2a behave as haplotypes, and that Alpowa clone 2 should be renamed PPO-D2b.

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Fig. 3. Chromosomal location of the newly identified PPO sequences. (A) Sequencespecific PCR amplification of Chinese Spring clones 4, 5, and 7 from Genomic DNA isolated from selected nullisomicetetrasomic and ditelosomic lines of T. aestivum Chinese Spring The primer set The PPOCln7F1/PPOCln7R1 set amplifies a 404 bp product from clone 7. The primer setPPOCln4F1/PPOCln4R1 primer set amplifies a 493 bp product from clone 4. The PPOCln5F1/PPOCln5R1 primer set amplifies a 275 bp product from clone 5. Lane identity is as follows: lane 1, Chinese Spring nullisomic 1A-tetrasomic 1D (N1A-T1D); lane 2, N1B-T1A; lane 3, N1D-T1A; lane 4, ditelosomic 2AS (DT2AS); lane 5, N2B-T2A; lane 6, DT2DS; lane 7, N3B-T2A; lane 8, N3D-T3A; lane 9, N3D-T3A; lane 10, N4A-T4D; lane 11, DT4BS; lane 12, N4D-T4B; lane 13, N5A-T5B; lane 14, N5B-T5A; lane 15, N5D-T5A; lane 16, N6A-T6B; lane 17, N6B-T6D; lane 18, N6D-T6A; lane 19, N7A-T7B; lane 20, N7B-T7A; lane 21, N7D-T7A; lane 22, Chinese Spring. M, Molecular size standards (750, 500, and 250 bp). (B) Polymorphic analysis of clones 2 and 5 in twenty-two wheat varieties. Sequence-specific primers for clones 2 and 5 were used in PCR to identify the presence and/or absence of Alpowa clone 2 and Chinese Spring clone 5 in each cultivar. The PPOCln5F1/PPOCln5R1 primer set amplifies a 275 bp product specific to clone 5, while the PPOCln2F1/PPOCln2R1 primer set amplifies a 275 bp product specific to Alpowa clone 2. Molecular size standard positions (750, 500, and 250 bp) shown at right. Lane identity is as follows: lane 23, Alpowa; lane 24, ‘UC1110’; lane 25, ‘CIMMYT-2; lane 26, ‘Rio Blanco’; lane 27, ‘IDO444’; lane 28, ‘IDO556’; lane 29, ‘Zak’; lane 30, ‘Louise’; lane 31, ‘Penewawa’; lane 32, ‘Weebil 1’; lane 33, ‘Jupateco 73S’; lane 34, ‘SS550’; lane 35, ‘Pioneer Variety 26R46’; lane 36, ‘P91193’; lane 37, ‘P92201’; lane 38, ‘Cayuga’; lane 39, ‘Caledonia’; lane 40, ‘Platte’; lane 41, ‘CO940610’; lane 42, ‘Tam 105’; and lane 43, ‘Jagger’.

3.5. Dynamics of gene expression of PPO genes in developing kernels RNA from the genes designated PPO-A1a, PPO-D1b, PPO-A2b, and PPO-D2b was detected in nascent seeds collected at each of the time points, while PPO-B2b was not detected at any of the time points. Gene PPO-A2b was by far the most active as measured by transcript level. At 2 dpa, gene PPO-A2b was responsible for virtually all of the detected PPO transcripts and at 9 dpa it was responsible for 200.7 of the estimated 252.9 PPO transcripts per ng cDNA (Table 2). While expression of PPO-A2b peaked at 9 dpa, expression of the other three genes peaked at 16 dpa (Fig. 4A). Expression levels of all four genes declined from 16 to 37 days (Fig. 4A); at 37 dpa PPO-A2b again accounted for virtually all of the detected PPO transcripts (Table 2). From these results we hypothesize that the levels of polyphenol oxidase protein synthesized in developing seeds largely are determined within about the first three weeks of seed formation, and that the level of polyphenol oxidase encoding mRNA (and subsequently protein) is determined by at least four distinct genes that are somewhat differentially regulated. A fifth gene, PPO-B2b, does not appear to be expressed in developing seed. Previous work by others has shown that for the PPO-A1a gene, protein levels closely follow transcript levels (AY596268 in Jukanti et al., 2006). Similar time courses for PPO activity and protein levels were also obtained by Anderson et al. (2006). We hypothesize that this

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Table 2 Estimated transcript copy number of wheat polyphenol oxidase genes per nanogram cDNA derived from developing seeds. Days post-anthesis 2 9 16 23 30 37 a b c

Number of copies of transcript from genea PPO-A1a

PPO-A2b

PPO-D1b

PPO-D2b

0.1 42.1 228.6 14.6 8.2 0.0

10.7 200.7 150.3 16.5 5.0 1.5

0.1 0.9 10.8 0.7 0.2 0.0

0.3 9.2 55.8 4.8 2.6 0.1

Total number of transcripts

% of transcripts from A genomeb

PPO-2c

11.1 252.9 445.5 36.5 15.9 1.6

96.8 96.0 85.1 85.0 82.5 92.0

98.3 83.0 46.3 58.3 47.4 99.5

Gene names indicate genome of origin (A or D), paralogous group (PPO-1 or PPO-2) and individual gene (A or B). The remainder of the transcripts were from the D genome. The remainder of the transcripts were from paralogous group PPO-1.

transcript/protein level association will likely hold true for the PPO2 gene family as well. In addition to these transcript quantitation results, a revisiting of results shown in Anderson et al. (2006) supports the conclusion that members of both PPO-1 and PPO-2 paralogous families are expressed as protein in developing kernels. Anderson et al. (2006) identified two distinct polypeptides of 60 and 62 kD. The peptides identified from the 60 kD sequence match those found in the previously identified PPO-D1 predicted gene product, (but not that of PPO-A1), whereas peptides from the 62 kD match those found in the predicted products of the PPO-A2a,b, PPOB2a,b, and PPO-D2a,b genes described here (Supplementary Fig. 1). Variation in expression levels of the groups of paralogous genes was estimated by comparing the mean expression of the two paralogous genes PPO-A1a and PPO-D1b to the mean expression of genes PPO-A2b and PPO-D2b (Fig. 4B). Over 80% of the PPO transcripts were from PPO-2 genes at 2, 9, and 37 dpa, while essentially equal amounts of transcripts were from PPO-1 and PPO-2 genes at 15, 23, and 30 dpa (Table 2). Over the 37-day period examined, the group 2 paralogous genes contributed on average 72.1% of the PPO gene transcripts present in the developing seeds. The average contributions of the orthologous A and D genes were estimated from the mean expression levels of genes PPO-A1a and PPO-A2b from the A genome, and D1b and D2b from the D genome. The A genome consistently contributed greater expression than the D genome at all of the measured time points (Fig. 4C). The difference in expression over the 37-day period ranged from 4.7- to 30.5-fold, with an average of 13.7-fold greater expression of genes from the A genome versus those from the D genome (Fig. 4C). The A genome contributed more than 80% of the PPO transcripts present in the developing seeds at each of the time points (Table 2). Averaged over the 37 day period examined, the A genome was responsible for 89.6% of the PPO gene transcripts in the developing seeds. 4. Discussion It is well established that PPO exists as a multigene family in wheat (Jukanti et al., 2004; Massa et al., 2007). Jukanti et al. (2004) identified two paralogous types, that of a ‘non-kernel’ type, thus named because of its absence in kernel tissue cDNA libraries, as well as a ‘kernel tissue’ type identified in developing seed libraries (2004). Two members of the kernel type have been genetically characterized from T. aestivum and their chromosomal locations identified. They are PPO-A1 identified by Sun et al. (2005) and PPOD1 identified by He et al. (2007). PPO-A1 and PPO-D1 appear to be orthologs based on sequence identity and chromosomal location. Massa et al. (2007) identified additional sequences within the kernel-specific group that were themselves divergent from the PPO-A1 and PPO-D1 genes (Fig. 3, Massa et al., 2007). Here, we have identified and characterized a new paralogous gene family distinct from the previously identified PPO-A1 and PPO-D1. This new family

consists of three orthologous members, which have been named PPO-A2, PPO-B2, and PPO-D2. The PPO-1 and PPO-2 gene families share many structural elements. Sequence identity analyses places them in closely-related phylogenetic groups. Also, the location of intron I is conserved both within and between PPO-1 and PPO-2 gene families (Fig. 1). This is in contrast to the outlying ‘non-kernel-type’ paralogous group, which contains no introns. The data thus point to a common origin for PPO-1 and PPO-2. The location of intron II is likewise conserved between the PPO-1 and PPO-2 groups. However intron II is missing from the PPO-A2a, PPO-A2b, and PPO-D1a sequences. This is intriguing from an evolutionary perspective. Based on comparison with the PPO-1 group, it appears as though the intron II was present in the ancestral kernel-specific sequence. Intron II appears to have been lost in the A genome or its diploid progenitor Triticum urartu. It also appears to have been lost in the D genome lineage that gave rise to PPO-D2a but was retained by the PPO-D2b progenitor. Intron II appears to fit the ideal criteria for loss as reported by Roy and Gilbert (2005). In their study of intron loss in 684 groups of orthologous genes from seven sequenced genomes, they concluded that introns at the 30 position that are in the phase zero position (i.e. does not interrupt a codon) are preferentially lost. Intron II of the PPO-2 orthologous genes fit these criteria. The presence of two divergent sequences at PPO-D2 is noteworthy. These results parallel those found by He et al. (2007) at the paralogous PPO-D1 gene. Hexaploid wheat is thought to be the result of more than one hexaploidization event (Giles and Brown, 2006; Talbert et al., 1998). The two D genome alleles could therefore have originated from divergent Aegilops tauschii progenitors. Introgression from a related Aegilops species is another possible explanation. Both PPO-D2a and PPO-D2b are well represented in a survey of 124 randomly chosen modern wheat varieties and breeding lines. Its prevalence in modern wheat germplasm suggests that their origin is either ancient or a recent introgression from another species under strong selection pressure (e.g. for disease resistance). For the first time, a set of five distinct members of the kernel-type PPO genes has been isolated in their entirety from a single cultivar of T. aestivum. This result was repeated in two cultivars, Chinese Spring and Alpowa. This has allowed us to demonstrate here, that in addition to the non-kernel PPO paralog, the kernel-type PPO gene cluster in fact contains a minimum of two paralogous gene families. Both kernel-type paralogous gene families are expressed from the A and D genomes (4 genes total) in developing kernels in the bread wheat cultivar Alpowa. The previously described genes PPO-A1 and PPO-D1 have been localized to the long arm of chromosomes 2A and 2D respectively. Here we demonstrate that the newly identified set of paralogous genes, here designated PPO-A2 and PPO-D2, are likewise localized to the long arm of chromosomes 2A and 2D. The PPO-B2 gene also has been identified and localized to chromosome 2B. However, unlike the

B. Beecher, D.Z. Skinner / Journal of Cereal Science 53 (2011) 371e378

Fig. 4. Transcript level developmental time course for each of PPO gene sequences now known to be expressed in developing wheat kernels. X-axis, time points (days post-anthesis) at which developing kernels were harvested and cDNA prepared as template. Y-axis, transcript level expressed as number of transcript copies/ng cDNA (logarithmic scale). (A) All four expressed PPO genes shown independently from one another. (B) Mean value of A genome PPO sequence expression (mean of PPO-A1a and PPO-A2b gene transcript levels) versus mean value of D genome PPO sequence expression (mean value of PPO-D1b and PPO-D2b gene transcript levels). (C) Mean value of PPO paralog 1 sequence expression (mean value of PPO-A1a and PPO-D1b gene transcript levels) versus mean value of paralog 2 sequence expression (mean value of PPO-A2a and PPO-D2b gene transcript levels).

four previously mentioned genes on the 2A and 2D genomes, PPOB2 expression was not detected in developing kernels of T. aestivum cv Alpowa. This is in agreement with the results of Jukanti et al. (2006), where expression of GenBank accession AY596269, which shows strongest sequence identity with PPO-B2, was not detected. Presence of a corresponding ‘PPO-B1’ gene can be inferred but has not yet been demonstrated, although the partial sequence in accession DQ889708 reported by Massa et al. (2007) is a strong

377

potential candidate for this gene. The PPO loci on Chromosome 2B may be non-functional or of limited function in a substantial portion of wheat cultivars. We did not observe expression from PPO-B2b, and the literature does not support a substantial role of chromosome 2B in PPO activity levels, although it has been mentioned in a few reports (Demeke et al., 2001; Fuerst et al., 2008; Watanabe et al., 2004). These data and interpretations are therefore in agreement with previous studies which localize PPO activity to the long arms of chromosomes 2A and 2D (Anderson et al., 2006; Chang et al., 2007; Demeke et al., 2001; He et al., 2007; Jimenez and Dubcovsky, 1999; Mares and Campbell, 2001; Raman et al., 2005; Simeone et al., 2002; Sun et al., 2005; Watanabe et al., 2004; Zeven, 1972; Zhang et al., 2005). Now that it is apparent that both the PPO-1 and PPO-2 genes contribute to seed PPO gene expression levels and are present on the same chromosomal arms, it seems likely that they are genetically linked. This would be consistent with the hypothesis that the two paralogs arose by a gene duplication event as proposed by Jukanti et al. (2004). This would be a situation analogous to that of the hardness locus found in some members of the Triticeae, where two linked genes contribute to a kernel textural phenotype (Beecher et al., 2001; Giroux and Morris, 1998). If they are indeed genetically linked, this has important implications for plant breeding efforts that strive to minimize PPO activity levels in modern wheat cultivars. The results of this study also indicate that at least two paralogous PPO genes, the previously described PPO-1, and the new PPO-2 genes described in this work, are expressed to a high degree in developing wheat seeds. The PPO-2 genes contributed over 70% of the PPO activity, based on gene transcript number; genes within the A genome were responsible for nearly 90% of the PPO transcripts. It should be noted that the cultivar Alpowa used in this study contains the PPO-D1b allele, which has been shown to be associated with higher kernel PPO activity levels (He et al., 2007). Surprisingly, the expression level of PPO-D1b was observed here to be a small fraction of that from the other expressed genes. It is therefore expected that the relatively major contribution toward PPO expression observed here will likely also be observed for cultivars containing the PPO-D1a allele as well. It should be pointed out that transcript levels are not a direct measure of the levels of the protein they encode. However previous work has shown that for the PPO-A1a gene, PPO protein levels can be correlated with transcript levels (Jukanti et al., 2006). If this holds true for these PPO-2 paralogs, this would indicate that they are the major contributor to PPO activity in the wheat kernel. However it is likely that both PPO-1 and PPO-2 gene families contribute substantially to wheat kernel PPO protein levels, and indeed a revisiting of the results of Anderson et al. (2006) supports this hypothesis. In this study, the levels of the P-62 kDa and P-60 kDa proteins (PPO-1 and PPO-2, respectively) appear to be present in similar levels based on visual observation of the gel (Anderson et al., 2006). It is also important to bear in mind that additional factors, namely substrate specificity and proteolytic activation, can influence the level of PPO activity observed. The degree to which the PPO-1 and PPO-2 gene family members behave in relation to these and other posttranslational factors is unknown and a subject for future study. In conclusion, we suggest that PPO activity in wheat kernels is determined by a complex array of orthologous and paralogous genes, each contributing transcripts, and therefore functional protein, to the total. The PPO-1 and PPO-2 orthologs present on the A genome together contribute far more transcript than those from either the B or D genome. The PPO-2 genes we have described in this work contribute the majority of the observed transcript levels. Therefore these newly-described PPO-2 genes, particularly PPO-A2, should be considered and addressed in any effort made to reduce PPO activity in wheat and food products derived from wheat.

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