Time course effects on primary metabolism of potato (Solanum tuberosum) tuber tissue after mechanical impact

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Abstract

An important feature relevant to potato (Solanum tuberosum) tuber quality management is the susceptibility to blackspot bruising caused by melanin production after exposure to severe mechanical stress. We analyzed the previously neglected nature and sequence of responses in primary metabolism and compared the cultivars ‘Afra’ and ‘Milva’ that differ in tissue elasticity and susceptibility to blackspot bruising. Mechanical stress application was highly standardized and differential formation of bruising damage and occurrence of small cracks in the outer tuber layers were demonstrated. Concomitant GC–TOF-MS based metabolite profiling revealed characteristic changes in central metabolism, namely in intermediates of the TCA cycle and linked parts of amino acid metabolism, with the clear exception of phenylalanine and tyrosine, the immediate precursor of melanin biosynthesis. We conclude that the initial metabolic responses and the initiation of blackspot formation are distinct, and that melanin biosynthesis is not driven by early precursor accumulation through activation of the aromatic amino acid biosynthesis pathway.

Introduction

Mechanical impact on potato tubers during harvest and subsequent handling not only causes external damage such as cracking and scuffing of the skin (Hughues, 1980), but can also result in a bluish-grey to black internal discoloration of the tuber tissue. This so-called blackspot bruising is hardly visible from the outside as it is located about 2 mm beneath the surface of the tuber in the region of the vascular ring. Blackspot bruising typically occurs within 1–3 d after mechanical impact (Burton, 1989, Molema, 1999) and is considered a significant impediment for the utilization of tuber harvests throughout the world as it severely influences consumer acceptance (Peters, 1996).

Several parameters and tissue features are known to be associated with the susceptibility of potato tubers to bruising damage, such as temperature, tissue toughness, turgor pressure, starch content, potassium content, location of the impact on the tuber, tuber age and storage duration (Kunkel and Gardner, 1959, Aeppli and Keller, 1979, Brook, 1996, McGarry et al., 1996, Peters, 1996, Bajema et al., 1998, McNabnay et al., 1999, Van Canneyt et al., 2006). Some of these parameters are known to relate to mechanical tuber properties like the transmission of impact energy and the tendency of the tissue to crack upon collision (McGarry et al., 1996). Therefore, failure properties after dynamic compression of potato tuber tissue can be used to characterize differences between potato cultivars with regard to bruising sensitivity and mechanical impact (Bajema et al., 1998). Furthermore, the severity of damage appears to depend on physiological and biochemical properties such as differences in specific metabolite levels or enzyme activities, such as the chemical oxidation reactions and intermediates that are directly involved in the internal discoloration.

Upon mechanical impact, cell membranes have been shown to be damaged and the enzyme polyphenol oxidase (PPO) and phenolic substrates that are normally separated by different subcellular compartmentation come into contact now. Furthermore, PPO activity exhibits a subcellular redistribution response 12 h after mechanical impact without apparent changes in the corresponding mRNA levels (Partington et al., 1999). The amino acid tyrosine is regarded as the most important substrate for PPO-catalyzed oxidative conversion. This reaction leads to the production of quinones, which subsequently react via a non-enzymatic reaction to the black pigment melanin (Corsini et al., 1992, Dean et al., 1993). High tyrosine content is known to correlate with a high susceptibility to internal discoloration. Other phenolic compounds such as chlorogenic acid or caffeic acid are known to be relevant but their contribution to blackspot formation is less clear (Dean et al., 1993, McGarry et al., 1996, Laerke et al., 2002). For example, the formation of iron-chlorogenic acid chelates has been suggested to contribute to the internal discoloration of potato tuber tissue (Putz, 1995).

In contrast to what is well known about the chemical reactions directly involved in blackspot formation, only little is known about the involvement of central metabolism, especially primary carbohydrate metabolism such as the respiratory metabolic pathways consisting of glycolysis and the subsequent tricarboxylic acid (TCA) cycle. Amino acid metabolism, and the time lag between mechanical impact and the onset of discoloration have also been largely neglected until now. The initial mechanical stress signal is expected to be linked to later biochemical responses via intermediate metabolic processes as earlier studies which were the incentive for this investigation demonstrated that respiratory activity increased significantly after mechanical stress application to potato tubers (Aeppli and Keller, 1980). Indeed, a correlation of this effect with formation of blackspot bruising also became apparent in our study.

Additional knowledge was generated by previous analyses of transcriptional regulation. This evidence points towards an intermediate metabolic reprogramming. Specifically, the transcriptional induction of ubiquitin, an indicator of protein degradation, and of phenylalanine ammonia-lyase, the key step of phenylpropanoid biosynthesis including chlorogenic acid production, have been linked to the mechanical impact response of potato tubers (Rickey and Belknap, 1991).

In the present study, we investigated the biochemical changes in primary metabolism using the metabolomic toolbox of GC–TOF-MS based metabolite profiling for time course analyses. For this, a well established profiling method (Erban et al., 2007) was applied which is dedicated to a comprehensive analysis of intermediates of primary carbon and nitrogen metabolism. Only limited information about secondary metabolism can be obtained by using the method applied here as most compounds of secondary metabolism have a far too large molecular weight to be analyzed and are not properly derivatized.

The study targets a time window after mechanical impact but before any visible damage and discoloration of the potato tuber tissue occurred. Care was taken to perform a differential tissue analysis by applying highly standardized mechanical impact and by comparing the mechanically stressed part of each tuber to an equivalent part of the same tuber, which was not exposed to the direct collision event. The observed changes in central metabolism were associated with the susceptibility and the extent of subsequent blackspot formation of two potato cultivars. These potato cultivars had been chosen according to their differential physical tuber properties, which are demonstrated to affect the mechanical transmission of the force of impact.

Section snippets

Plant material

Potato (Solanum tuberosum cv. ‘Afra’ and cv. ‘Milva’) tubers were manually harvested in September and October 2007 from a field used for commercial potato tuber production near Niedergörsdorf (51°59′N, 13°0′S) in the region of southern Brandenburg, Germany. Harvested tubers were stored at 4 °C without a sprouting inhibitor. In a previous study (Praeger et al., 2010) physiological properties of the tubers were monitored several times during the storage period starting from harvest in autumn 2007

Susceptibility to blackspot bruising and cracking

A tuber drop-test was carried out to determine the susceptibility to blackspot bruising and cracking of ‘Afra’ and ‘Milva’ potatoes (Table 1). Both cultivars were more susceptible to blackspot bruising when tubers were dropped on their side as compared to the apical end. This was not only apparent from the percentage of tubers that developed blackspots as was determined two days after impact, but also by the size of the resulting blackspots. ‘Afra’ tubers were obviously much more susceptible to

Discussion

An immense variability exists between potato cultivars in the susceptibility to potato tuber damage like blackspot bruising. The chemical reactions that lead to the discoloration of tuber tissue after impact are well described, and it is known that metabolic precursors of primary metabolism are involved in melanin biosynthesis. However, hardly anything is known about how primary metabolism of the potato tuber reacts to mechanical impact specifically during the interval before blackspot bruising

Conclusions

Our study demonstrated that upon collision of potato tubers, specific metabolic changes are induced. These changes predominantly include intermediates of primary metabolism. Especially respiratory metabolism is increased, including the up-regulation of the levels of specific TCA cycle intermediates. Concomitantly, amino acids that are derived from TCA cycle precursors are depleted. The amplitude of the metabolic changes depended on the cultivar being investigated. It appeared that identical

Acknowledgements

The authors thank Anke Langer and Hildegard Hopp for help with the preparation of some of the material. This research obtained financial support from the German Federal Ministry of Education and Research (BMBF, QUANTPRO INNOX “Innovative diagnostic tools to optimize potato breeding: systemic analysis of cellular processes and their relation to plant internal oxygen concentrations” PTJ-BIO/Wir/0101-31P4295).

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    1

    These authors contributed equally to this work.

    2

    Present address: Metanomics GmbH, Tegeler Weg 33, 10589 Berlin, Germany.

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