Polyphenol Oxidase Gene Expression Research Articles

Reduced polyphenol oxidase gene expression and enzymatic browning in potato (Solanum tuberosum L.) with artificial microRNAs

BackgroundPolyphenol oxidase (PPO), often encoded by a multi-gene family, causes oxidative browning, a significant problem in many food products. Low-browning potatoes were produced previously through suppression of PPO gene expression, but the contribution of individual PPO gene isoform to the oxidative browning process was unknown. Here we investigated the contributions of different PPO genes to total PPO protein activity, and the correlations between PPO protein level, PPO activity and tuber tissue browning potential by suppression of all previously characterized potato PPO genes, both individually and in combination using artificial microRNAs (amiRNAs) technology.ResultsSurvey of the potato genome database revealed 9 PPO-like gene models, named StuPPO1 to StuPPO9 in this report. StuPPO1, StuPPO2, StuPPO3 and StuPPO4 are allelic to the characterized POTP1/P2, POT32, POT33 and POT72, respectively. Fewer ESTs were found to support the transcriptions of StuPPO5 to StuPPO8. StuPPO9 related ESTs were expressed at significant higher levels in pathogen-infected potato tissues. A series of browning phenotypes were obtained by suppressing StuPPO1 to StuPPO4 genes alone and in combination. Down-regulation of one or several of the PPO genes did not usually cause up-regulation of the other PPO genes in the transgenic potato tubers, but resulted in reduced PPO protein levels. The different PPO genes did not contribute equally to the total PPO protein content in the tuber tissues, with StuPPO2 accounting for ~ 55% as the major contributor, followed by StuPPO1, ~ 25-30% and StuPPO3 and StuPPO4 together with less than 15%. Strongly positive correlations between PPO protein level, PPO activity and browning potential were demonstrated in our analysis. Low PPO activity and low-browning potatoes were produced by simultaneous down-regulation of StuPPO2 to StuPPO4, but the greatest reduction occurred when StuPPO1 to StuPPO4 were all suppressed.ConclusionStuPPO1 to StuPPO4 genes contributed to browning reactions in tuber tissues but their effect was not equal. Different PPO genes may be regulated independently reflecting their diversified functions. Our results show that amiRNAs can be used to suppress closely related members of highly conserved multi-gene family. This approach also suggests a new strategy for breeding low-browning crops using small DNA inserts.

Polyphenol Oxidase (PPO) in Early Stage of Browning of Phalaenopsis Leaf Explants

Explant browning is one of the most common problems during the early stage of tissue culture. The aim of this study was to explore the roles of polyphenol oxidase (PPO, EC 1.10.3.2) in the browning process of leaf explants of Phalaenopsis (Doritaenopsis Queen Bee “Red Sky”) under a routine culture condition. Along with the observation of the time course of browning and PPO distribution, PPO gene expression in leaf explants were investigated. In addition, the expression and activity of PPO were also examined. This work provides some insights into further understanding the relationship between PPO activation and explant browning.

Maturity and cooling rate affects browning, polyphenol oxidase activity and gene expression of ‘Yali’ pears during storage

Browning is the main physiological disorder of ‘Yali’ pear (Pyrus bretschneideri Rehd) during storage. In this study, the relationships between browning development in fruit from different harvest dates, and cooled either rapidly or slowly, with polyphenol oxidase (PPO) activity and isozymes, and PPO gene expression has been investigated. Development of browning was highest in late-harvest fruit in both core and flesh tissues and was higher in rapidly cooled than slowly cooled fruit. Mid-harvest fruit had the lowest browning incidence and PPO activity of core tissue was higher than in flesh and seeds, while the peak of PPO activity in mid-harvest fruit was the lowest. Six PPO isoenzymes were detected in fruit, three bands A, B and E in flesh and core tissues, three bands C, D and F in the seeds. The intensity of PPO isoenzyme staining of bands A and B in pulp and core was similar to that of PPO activity and browning incidence. PPO gene expression increased and then decreased in core tissues. Trends of expression were similar to those of PPO activity. Rapid cooling promoted the expression PPO. The results suggest PPO plays an important role in ‘Yali’ pear browning during storage.

Molecular Cloning and Expression of Polyphenoloxidase Genes from the Mushroom, Agaricus bisporus

The polyphenoloxidase (PPO) is the key enzyme considered to be responsible for mushroom browning. By using homology cloning and rapid amplification of cDNA ends (RACE), two new PPO genes and the corresponding cDNA were identified from the fruit bodies of Agaricus bisporus (AbPPO3 and AbPPO4, GenBank accession nos. GU936494 and GU936493, respectively). The genomic DNA sequences of AbPPO3 and AbPPO4 are 2 080 and 2 189 bp in length, respectively, encoding putative polypeptides of approximately 66 and 68 kDa. The deduced amino acid sequences show characteristic features of two copper-binding domains conserved in the type III copper proteins including fungal polyphenol oxidases. Sequence comparisons indicate that AbPPO3 and AbPPO4 present 55.3% similarity to each other (48% identity). We also obtained more than 1.5-kb long sequences upstream of the start codon of the AbPPO3 and AbPPO4 and recognized them as their respective putative promoters. Analyses of the two PPO promoter regions show that they contain abundant cis-acting elements which are probably responsible for anaerobic induction, light, wound, stress, and auxin response. Semi-quantitative RT-PCR results indicate that AbPPO3 and AbPPO4 were highly expressed in the mature fruit bodies and up-regulated after 2-d storage of mushroom. These results suggest that AbPPO3 and AbPPO4 may play roles in A. bisporus, browning and pigmentation during development and postharvest storage and the elements in promoters may act as regulatory elements for the inducible expression of AbPPO3 and AbPPO4. The successful cloning and expression analysis of AbPPO3 and AbPPO4 warrant a further investigation on the structure and function of A. bisporus PPO which points to the possible targets for genetic manipulation.

Alternative splicing in the coding region of Ppo-A1 directly influences the polyphenol oxidase activity in common wheat (Triticum aestivum L.)

Polyphenol oxidase (PPO) plays a crucial role in browning reactions in fresh and processed fruits and vegetables, as well as products made from cereal grains. Common wheat (Triticum aestivum L.) has a large genome, representing an interesting system to advance our understanding of plant PPO gene expression, regulation and function. In the present study, we characterized the expression of Ppo-A1, a major PPO gene located on wheat chromosome 2A, using DNA sequencing, semi-quantitative RT-PCR, PPO activity assays and whole-grain staining methods during grain development. The results indicated that the expression of the Ppo-A1b allele was regulated by alternative splicing of pre-mRNAs, resulting from a 191-bp insertion in intron 1 and one C/G SNP in exon 2. Eight mRNA isoforms were identified in developing grains based on alignments between cDNA and genomic DNA sequences. Only the constitutively spliced isoform b encodes a putative full-length PPO protein based on its coding sequence whereas the other seven spliced isoforms, a, c, d, e, f, g and h, have premature termination codons resulting in potential nonsense-mediated mRNA decay. The differences in expression of Ppo-A1a and Ppo-A1b were confirmed by PPO activity assays and whole grain staining, providing direct evidence for the influence of alternative splicing in the coding region of Ppo-A1 on polyphenol oxidase activity in common wheat grains.

Two polyphenol oxidases are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple

Polyphenol oxidase (PPO), a copper-containing metalloprotein, catalyzes the oxidation of phenolics to quinones which make brown pigments in wounded tissues. Because the phenomena decrease fruit quality, PPO has been regarded to be a critical enzyme in food technology. In the course of expressed sequence tags (ESTs) analysis of the Fuji apple ( Malus domesticus Borkh.), we identified two partial PPO cDNA clones; F114 corresponded to the previously isolated Granny Smith apple pAPO5 (Plant Mol. Biol. 27 (1995) 429), while F226 was a new clone. Using F226 as a probe, we isolated a full length PPO clone, pMD-PPO2, from a cDNA library prepared from young fruits of the Fuji apple. The deduced amino acid sequences of pMD-PPO2 and pAPO5 share 55% identity, and display a high degree of sequence identity (43–58%) with previously identified PPO from various species. RNA gel blot analysis using gene-specific probes showed that two apple PPO genes display unique patterns of expression in a tissue- and developmental-specific manner. In the process of flower development, the APO5 transcript was detectable only at the post-anthesis stage. In contrast, MD-PPO2 was expressed in all stages of flower development, with the abundance of mRNA being the highest at the pre-anthesis stage and then receding as the flower developed. Both genes are expressed in the early stages of fruit development. The expression was dramatically reduced as the fruit ripened. In leaf tissue, the APO5 gene was highly expressed in young and immature leaves, while MD-PPO2 was transcriptionally more active in both immature and mature leaves. Upon wounding, APO5 was significantly induced in leaves and fruits, whereas the level of MD-PPO2 mRNA was not affected by mechanical damage. Thus, it appears that two Fuji PPO genes are differentially expressed during vegetative and reproductive development and in response to wounding in the Fuji apple plants. The possible molecular mechanism of differential regulation of PPO gene expression in apple plant and its physiological significance are discussed.

Induction of apple scald by anaerobiosis has similar characteristics to naturally occurring superficial scald in ‘Granny Smith’ apple fruit

Abstract Scald, which can be induced by anaerobiosis (N2-induced), may involve similar physiological and biochemical mechanisms to those which contribute to naturally occurring superficial scald. To investigate this hypothesis, Granny Smith apples were harvested weekly from four orchards and stored in air for 16 weeks at 0°C, before being ventilated with N2 for up to 4 days, or held in air at 20°C. Following N2 treatment, scald developed within a few minutes of transfer to air and its severity was positively correlated with duration of N2 treatment. Susceptibility to induction of scald with N2 decreased through the harvest season and increased with length of refrigerated storage. Following N2 treatment, the levels of α-farnesene, conjugated triene (CTH) and OD200 (Abs200) were significantly lower than in fruit held continuously in air. This effect was more apparent in the early than late harvest fruit. RNA gel blot analysis of polyphenol oxidase (PPO) was conducted on selected samples. Expression of PPO was very low while fruit were held in N2, suggesting that the regulation of PPO gene expression is dependent on oxygen. Once fruit were removed to air, browning occurred almost immediately, too rapidly for the initial development of browning symptoms in scald to be attributed to increased PPO gene expression. We conclude that anaerobically induced scald development has similar characteristics of naturally occurring superficial scald and in both cases elevated PPO gene expression may not be associated with the initial development of symptoms.

Antisense Expression of Polyphenol Oxidase Genes Inhibits Enzymatic Browning in Potato Tubers

Spoilage caused by post–harvest enzymatic browning is a problem of considerable importance to food growers, processors and retailers. Here we show that antisense inhibition of polyphenol oxidase (PPO) gene expression abolishes discoloration after bruising of potato tubers in individual transgenic lines grown under field conditions. Using appropriate promoters to express antisense PPO RNA, melanin formation can be specifically inhibited in the potato tuber. This lack of bruising sensitivity in transgenic potatoes, and the absence of any apparent detrimental side effects open the possibility of preventing enzymatic browning in a wide variety of food crops without resorting to treatments such as heating or the application of antioxidants.

Organisation of the tomato polyphenol oxidase gene family.

We report the isolation and characterization of seven nuclear genes encoding polyphenol oxidase (PPO) in tomato (Lycopersicon esculentum cv. VFNT Cherry). The seven genes (PPOs A, A', B, C, D, E and F) fall into three structural classes (I, II, and III) based on Eco RI and Hind III restriction fragment length polymorphisms (RFLP). RFLP mapping and PFGE analysis demonstrated that the genes reside on chromosome 8, and may be clustered within a 165 kb region. Phage insert mapping demonstrated PPO E and PPO F (both class III), and PPOs B, D and A (classes I, II and I respectively) are grouped within separate 12.4 kb clusters. The complete nucleotide sequence was determined for each gene. Comparison to cDNAs revealed that the PPOs lack introns. A transcript of about 2 kb is expected for each PPO. Each PPO possesses a region encoding a transit peptide characteristic of polypeptides targeted to the thylakoid lumen. Predicted precursor polypeptides range in mass from 66 to 71 kDa and predicted mature polypeptides range from 57 to 62 kDa. All the PPOs encode two putative copper-binding sites characteristic of bacterial, fungal and mammalian tyrosinases. Five of the seven PPOs possess divergent DNA sequences in their 5' promoter regions. These flanking sequence differences may regulate the differential expression of PPO genes.