Abstract
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.
Highlights
Polyphenol oxidase (PPO), often encoded by a multi-gene family, causes oxidative browning, a significant problem in many food products
A previous study based on RNA Northern blot analysis revealed that POTP1 and POTP2 genes were expressed mainly in potato leaves and flowers, POT32 and POT33 mRNA were detected mainly in tubers with the POT32 being the major form throughout tuber development, and POT72 gene was mainly expressed in the roots [1]
Genome-wide survey of PPO-like gene models in Solanum tuberosum A genome-wide search of the recent S. tuberosum whole genome database in the US Joint Genome Institute reveals 9 PPO-like gene models, tentatively named StuPPO1 to StuPPO9 in this report (Table 1, Additional file 1)
Summary
Polyphenol oxidase (PPO), often encoded by a multi-gene family, causes oxidative browning, a significant problem in many food products. Several reports have described reduced browning reaction in crops by suppression of PPO gene expression using transgenic transformation with PPO gene fragments in configurations such as sense, antisense or double-stranded RNA [4,5,6,7,8,9,10]. Those approaches functioned by establishing an RNA silencing mechanism guided by a population of heterogeneous small interfering RNAs (siRNAs) [11]. A previous study based on RNA Northern blot analysis revealed that POTP1 and POTP2 genes were expressed mainly in potato leaves and flowers, POT32 and POT33 mRNA were detected mainly in tubers with the POT32 being the major form throughout tuber development, and POT72 gene was mainly expressed in the roots [1]
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