Visual Detection of Calcium by GBHA Staining in Bitter Pit-affected Apples

Abstract

Bitter pit is a physiological disorder of apple fruits apparently caused by a localised calcium deficiency or imbalance in fruits associated with low levels of calcium in the flesh. A new, highly selective method using glyoxal bis(2-hydroxyanil, GBHA) was tested to reveal the presence of calcium within the fruit as a red stain. Water-soluble and insoluble calcium was analysed by capillary electrophoresis and atomic absorption spectroscopy in pitted regions, adjacent sound areas and pulp from sound apples. Both methods, selective calcium staining and mineral analysis, showed that calcium accumulates in the pitted areas of apples affected by bitter pit. However, in mechanically inflicted wounds, the pulp of the apple was heavily stained but not the corresponding fingerprint, indicating a similar mechanism of insoluble calcium accumulation but a different distribution of soluble calcium compared to the pits.Keywords: apple, bitter pit, calcium staining, glyoxal bis(2-hydroxyanil) INTRODUCTIONThe browning of plant tissues is a manifestation of mechanical damage, certain diseases and physiological disorders, senescence and a hypersensitive reaction induced by pathogenic microorganisms. It is generally believed that the process of browning is due to the loss of cell compartmentalisation and involves the oxidation of polyphenolic compounds by polyphenol oxidase, resulting in the production of polymerised melanin-like pigments (Vaughn and Duke, 1984). Calciumrelated physiological disorders in apples and other fruits also induce corky brown tissues in the fruit pulp. Bitter pit is a physiological disorder of apples (Malus x domestica Borkh) that has been related to calcium deficiency in the fruit (Ferguson and Watkins, 1989). The symptoms generally arise in stored fruit after harvest. Economic losses can be quite substantial since the affected apples cannot be sold as a fresh commodity and must be removed by hand. Many studies have shown that the calcium nutrition of horticultural crops has an important influence on fruit quality (Neilsen and Neilsen, 2003). Indeed, several physiological disorders in the storage organs of fruits and vegetables are related to inadequate calcium supply (Shear, 1975).The translocation of calcium in higher plants is mainly restricted to xylem sap, following the unidirectional transpiration stream (Kirkby and Pilbeam, 1984), unlike K + and Mg 2+ (and N and P), which are easily transported throughout the phloem (Bergmann, 1992;Tagliavini et al., 2000). Once * To whom correspondence should be sent J. Val et al2 incorporated within the leaf, calcium does not recycle, even under conditions of deficiency (Hanger, 1979). Calcium moves into the plant in ionic form or is complexed with malic or citric acids (Bradfield, 1976;Vang-Petersen, 1980). As pointed out by Zocchi and Mignami (1995), calcium is only slightly mobile in the xylem. Thus, it can reach high concentrations in transpiring tissues (leaves) through water mass flow, but is relatively scarce in low transpiring tissues such as fruit, seed...

‘Honeycrisp’ Bitter Pit Response to Rootstock and Region under Eastern New York Climatic Conditions

“…Most Ca 2+ in fruit tissue, between 60 and 75%, is bound to the cell wall. More Ca 2+ binding to the cell wall is consistent with the finding that BP-damaged tissues have more Ca 2+ than the surrounding healthy tissues [ 31 , 32 ]. In agreement with this statement and previous studies [ 3 , 33 ], we found a high and negative correlation between peel Ca 2+ concentration and BP incidence after storage for all three rootstock categories and two regions.…”

Mechanisms regulating apple cultivar susceptibility to bitter pit

“…Accordingly, fruit tissue with BP symptoms may have more cortical Ca 2+ than sound fruit (Saure, 2005). More Ca 2+ binding to the cell wall is consistent with the finding that BP-damaged tissues actually have more Ca 2+ than the surrounding healthy tissues, mostly in a water-insoluble form (Steenkamp et al, 1983;Val et al, 2008). Other nutrients, in addition to Ca 2+ , have been suggested to play a role in BP development in apple fruit, and commercial apple growers frequently rely on the ratio of these nutrients to Ca 2+ as a guide to predict fruit susceptibility to BP.…”

“…In this case, the water-soluble pectin fraction could also contribute to the water-soluble Ca 2+ content in the fruit tissue. The Ca 2+ loosely bound to short pectin chains that comprise the water-soluble pectin fraction may be exchangeable (Val et al, 2008;Caffall and Mohnen, 2009) and supply Ca 2+ to the cell as needed, reducing fruit tissue susceptibility to BP. On the other hand, long deesterified pectin chains that represent the water-insoluble fraction have been suggested to form strong interactions with Ca 2+ , making this ion unavailable to other cellular functions and increasing fruit susceptibility to BP (Ralet et al, 2001;Caffall and Mohnen, 2009;De Freitas et al, 2010).…”

Bitter pit in apples: pre- and postharvest factors: A review

“…Bitter pitted fruit have more water insoluble Ca than water soluble Ca fraction. However, in unaffected fruit there is no difference in concentrations between these two Ca fractions (Val et al, 2008).…”