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Relationship between total peroxidase activity and susceptibility to black point in mature grain of some barley cultivars

Maria Sulman1, Glen Fox1, Abdulla Osman1, Andy Inkerman1, Peter Williamson2, and Miriam Michalowitz2

1Barley Quality Laboratory, Farming Systems Institute, Toowoomba, Queensland
Pathology, Farming Systems Institute, Toowoomba, Queensland


Total peroxidase activity was measured in mature barley kernels of varieties showing high levels of resistance to black point (WA5034, WADH14497, Harrington) as well as in cultivars with low levels of resistance (WI2976, Schooner Tallon). Two hulless varieties (Namoi and SB85744) were also included in this study. Peroxidase was extracted from both clean and black point infected kernels in a sodium phosphate buffer at pH 7.0. Total peroxidase activity was assayed with ferulic acid at three different pH levels: 5.5, 7.0 and 8.5.

Results indicate that there was no a direct correlation between total peroxidase activity and the level of black point in the cultivars analysed. Barley varieties that are at opposite ends in the black point susceptibility scale like Harrington and WA5034 (tolerant) and WI2976, Schooner and Tallon (susceptible) exhibited very similar levels of total peroxidase activity. This suggests that there is sufficient peroxidase in mature barley kernels of all the varieties tested to cause black point and that differences in substrate or hydrogen peroxide may be the factor that distinguishes between resistance from susceptibility. Black point is initiated before grain maturity and it might be that peroxidase levels at these earlier developmental stages have differing levels than at maturity. However, it is still likely that peroxidases are involved in the biochemical mechanism that causes black point.


Black point of barley (Hordeum vulgare) is manifested by a black-brown discolouration at the germ end of otherwise healthy grain. Research on black point was initially focused on the assumption that this discolouration was the result of a saprophytic infection (Waldron 1934, Machacek and Greaney 1938, Southwell et al.1980). Black point symptoms were often described in relation to the mycelia density in the tissues affected and were mainly associated with the presence of Alternaria alternata (Rees et al. 1984, Conner and Kuzyk 1988, Cromey and Mulholland 1988). More recent studies suggest no direct association between black point and fungal infection (Jacobs and Rabie 1987, Basson et al. 1990, Ellis et al. 1996). Williamson (1997) found no direct association between infection with Alternaria alternata and black point symptoms. He also did not recognise a greater density of hyphae of Alternaria alternata in discoloured tissue of inoculated wheat kernels at the embryo end of the grain.

Black point is more likely to be an oxidised phenol resulting from the biochemical disruption of the ripening process brought about by stressful conditions (Williamson 1997). Williamson (1997) assayed for peroxidase enzymes and reported a relationship between black point susceptibility in wheat and the presence of peroxidase isoenzymes. The peroxidase and the phenols necessary for the development of black point symptoms are also components of barley grains (Cochrane 1994). Peroxidase plays an important role in stress related processes (Breda et al. 1993) and is responsible for browning in most damaged tissues. Cochrane (1994) also demonstrated that endogenous hydrogen peroxide could be produced by barley germ aleurone cells, which is essential for peroxidation to take place.

Regnier and Macheix (1996) measured peroxidase activity in a black point susceptible durum cultivar and a moderately resistant cultivar. Peroxidase activity was measured post anthesis and was repeated every two days until kernel maturity. Peroxidase activity was reported to be the highest in the susceptible durum cultivar prior to grain maturity. No differences in enzymatic activity between the resistance and susceptible durum varieties were observed at harvest.

The objectives of this study were: i ) to develop a method to measure total peroxidase activity in the germ end using natural substrates, ii ) to investigate the correlation between cationic, anionic isoenzymes and black point susceptibility in barley cultivars, iii ) to compare total peroxidase activity in clean and black point barley kernels.

Materials and Methods

Barley samples

Barley cultivars were grown under a high humidity environment at Hermitage Research Station, DPI, from June to December 2000. Cultivars with the highest (WA5034, WADH14497, Harrington) and lowest level of resistance (WI2976, Tallon, Schooner) to black point were chosen from the 90 barley genotypes screened for resistance to black point at Hermitage Research Station (Sulman et al. 2001). Two hulless cultivars, Namoi and SB8574, were also included.

Peroxidase extraction

De-husked barley germ ends (25 mg) were dissected from mature kernels, crushed and incubated at 25oC for 1 hr 40 mM sodium-phosphate buffer at pH: 7.0 containing 1% (v/v) Triton X-100 and 0.5 M KCl. Sucrose (25 mM) was added and the homogenate was centrifuged at 5500 g at 4oC for 10 min. The supernatant was stored at 25oC.

Total peroxidase measurements

Total peroxidase activity was measured using a reaction mixture consisting of 2.85 ml of 0.04% (w/v) ferulic acid and 4% H2O2. The reaction was initiated by adding 100 uL of the peroxidase extract and allowing it to react for 1 min at 25oC. Abs/min was measured at 350 nm for 1 min in the linear range of a Shimadzu UV- 250IPC spectrophotometer (absorbence 0.3). The concentration of the substrates at which maximum enzyme activity occur (ferulic acid 0.04 and H2O2 4%) was determined experimentally by maintaining the concentration of one substrate constant while varying the concentration of the other.

Peroxidase activity was measured at three different pH levels : 5.5, 7.0 and 8.5. A range of buffers, maleate, citrate, and Tris were used at pH 5.5, 7.0 and 8.5 respectively. The suitability of a range of substrates (catechol, catechin and ortho-dianisidine) for measuring total peroxidase activity was established during method development. The total peroxidase activity values reported are the average of three days of measurements.

Peroxidase units

Total peroxidase activity was reported in arbitrary units (U/g germ). One unit is the amount of enzyme that gives 0.1 abs at 25oC in 3 ml of reaction mixture.

Results and discussion

Method development:

Most of the germ end peroxidases were extracted in 1 hour. A linear relationship between the rate of reaction and the amount of peroxidase extracted in the reaction mixture (r2 = 0.988) was found. Enzyme activity was measured at increasing concentrations of peroxidase.

In addition, the maximum absorbance of the product formed in the peroxidase reaction was 350 nm. This wavelength was determined by scanning the reaction mixture after 1 min of adding the peroxidase extract over a wide wavelength range (200-600 nm). In regards to substrate, O-dianisidine proved to be a very effective substrate as only one product was formed during the peroxidase reaction. However, this substrate was not employed in the assay due to its mutagenic and carcinogenic nature. Catechol and catechin were not used as substrates in the final tests because two products were formed after adding the peroxidase extract to the reaction mixture. Scans of the solution containing the buffer and peroxidase showed two peaks in the ultraviolet range. Ferulic acid proved to be suitable for measuring total peroxidase activity for many reasons, it forms one product during the enzymatic reaction, is a natural substrate present in barley kernels and is not toxic.

The total peroxidase activity measurements at pH: 5.5, 7.0 and 8.5 were significantly different between the barley cultivars analysed (Table 1). The results showed that the optimum pH for peroxidase reaction was 7.0. The anionic isoenzymes were much more active than the cathodic isoenzymes. This may be explained by the fact that isoenzymes with PI 8.25 and 8.55 increased in activity at the end of grain filling period (Cochrane et al 2000).

No direct correlation was found between the amount of peroxidase extracted from mature barley kernels and the level of black point in the cultivars analysed. Genotypes that exhibited high levels of resistance to black point (Harrington and WA5034) displayed similar levels of total peroxidase activity to the genotypes with low levels of resistance to black point WI2976, Schooner, and Tallon. Regmier and Macheix (1996) reported similar results in durum wheat where they observed no differences in the level of peroxidase activity in mature grain of a moderate resistant and susceptible durum cultivar. Although, they assayed for peroxidase using the whole durum kernel and no attempt was made to measure peroxidase activity in the tissue affected by black point. In addition, Regnier and Macheic (1996) made no comparison between the levels of peroxidase extracted from clean and black pointed kernels.

The level of peroxidase extracted from barley kernels exhibiting black point symptoms and symptomless kernels was significantly different (p< 0.001) (Table II). For most cultivars, the level of peroxidase extracted from the black point kernel was lower than the amount of enzyme present in the symptomless kernels.

Table 1. Total peroxidase activity and standard error of some mature barley kernels exhibiting black point symptoms (b.p) and symptomless (clean).


Total Peroxidase Activity (U/gr germ)

PH 5.5

PH 7.0

PH 8.5








6130 (0.68)

3870 (0.50)

10110 (0.91)

6760 (0.70)

6600 (0.75)

4380 (0.39)


3960 (0.11)

3830 (0.36)

6930 (0.15)

7690 (0.73)

4490 (0.16)

5340 (0.60)


4290 (0.08)

4880 (0.47)

8070 (0.13)

8600 (0.89)

7370 (0.17)

7010 (0.29)


5460 (0.65)

4770 (0.34)

9460 (0.87)

8670 (0.35)

7660 (0.79)

7210 (0.31)


5050 (0.36)

4100 (0.58)

8500 (0.57)

7400 (0.83)

5690 (0.45)

5210 (0.63)


5551 (0.39)

3930 (0.61)

10270 (0.51)

8110 (1.03)

6990 (0.09)

6260 (1.04)




5930 (0.47)


4270 (0.37)



3880 (0.35)


6780 (0.33)


5830 (0.49)


Table 2. Statistical analysis



Wald / d.f.

Chi-sq prob









Type (clean-b.p)





There was not a direct correlation between level of susceptibility to black point and total peroxidase activity measured at the germ end of mature barley kernels at any pH measured (5.5, 7.0 and 8.5). This suggests that the level of peroxidase in mature barley kernel of all varieties analysed is sufficient to cause black point and that differences in substrate or hydrogen peroxidase may be the factor that distinguishes between resistance and susceptibility. Black point symptoms occur before grain maturity and it might be that peroxidase levels at these earlier developmental stages have differing levels than at maturity.

While there was not a strong relationship between the level of peroxidase and black point susceptibility in the genotypes assayed in this study, initial studies have indicated that particular phenolic acids, including ferulic acid and p-coumaric acid, significantly increase in black point tissue in barley (unpublished data). Further work will continue to investigate the relationship between particular peroxidase isozymes, total peroxidase activity and phenolic acid levels at stages of grain development post anthesis to confirm the role these components have on the development of black point. In particular, studies will include genotypes with higher levels of resistance to black point as identified in Sulman et al. (2001).


The authors wish to thank the Grains Research and Development Corporation for their financial support. Kerry Bell is acknowledged for the statistical analysis. Ross Ferguson, Karen Onley and Andrew Skerman are also acknowledged for their contributions.


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