Dhurrin synthesis in sorghum is regulated at the transcriptional level and induced by nitrogen fertilization in older plants

Abstract

The content of the cyanogenic glucoside dhurrin in sorghum (Sorghum bicolor L. Moench) varies depending on plant age and growth conditions. The cyanide potential is highest shortly after onset of germination. At this stage, nitrogen application has no effect on dhurrin content, whereas in older plants, nitrogen application induces an increase. At all stages, the content of dhurrin correlates well with the activity of the two biosynthetic enzymes, CYP79A1 and CYP71E1, and with the protein and mRNA level for the two enzymes. During development, the activity of CYP79A1 is lower than the activity of CYP71E1, suggesting that CYP79A1 catalyzes the rate-limiting step in dhurrin synthesis as has previously been shown using etiolated seedlings. The site of dhurrin synthesis shifts from leaves to stem during plant development. In combination, the results demonstrate that dhurrin content in sorghum is largely determined by transcriptional regulation of the biosynthetic enzymes CYP79A1 and CYP71E1.

Figure 1

The biosynthetic pathway for the cyanogenic glucoside dhurrin in sorghum.

Figure 2

The effect of seedling age on cyanide potential in sorghum. The cyanide potential is shown per plant (A) and per milligram of plant material (B; fresh weight). Seeds were imbibed in water for 3 h, and seedlings were grown for the time periods indicated. Cyanide potential in the whole plant except roots was measured as described in “Materials and Methods.”

Figure 3

The effect of seedling age on activity and amount of CYP79A1 and CYP71E1 in sorghum microsomes. A, Enzyme activity per milligram of fresh plant material; B, enzyme activity per milligram of microsomal protein; and C, western-blot analysis of the content of CYP79A1 and CYP71E1 with equal amounts of microsomal protein applied to each lane. Microsomes were prepared from whole seedlings except roots. The combined activity of CYP79A1 and CYP71E1 was measured with Tyr as substrate, and the activity of CYP71E1 was measured with (Z)-p-hydroxyphenylacetaldoxime as substrate. The cyanohydrin formed as the final product of the enzymatic reactions was hydrolyzed in alkali and cyanide release measured as described in “Materials and Methods.”

Figure 4

The effect of seedling age on mRNAs encoding CYP79A1 and CYP71E1 in sorghum. Total RNA was isolated and analyzed by northern blotting. Equal amounts of RNA were applied to all lanes as measured by ethidium bromide staining. The blots were hybridized with the CYP79A1 and the CYP71E1 probes as indicated.

Figure 5

The effect of nitrate on cyanide potential in sorghum plants at different developmental stages. A and B, Seeds were imbibed in water for 3 h, and seedlings were grown for the time periods indicated in water (⋄—⋄), 25 mm KCl (○—○), or 25 mm KNO₃ (×—×). C and D, Plants were grown for 35 d in soil. Watering was then continued with H₂O (⋄—⋄) or changed to 25 mm KCl (○—○) or 25 mm KNO₃ (×—×) for the time period indicated. The cyanide potential in the whole plant except roots was measured as described in “Materials and Methods.”

Figure 6

The effect of nitrate on induction of CYP79A1 and CYP71E1 catalytic activity and cyanide potential in different parts of 5-week-old sorghum plants. A, Microsomes were prepared from different plant parts, and their ability to convert ¹⁴C-labeled l-Tyr to p-hydroxymandelonitrile was analyzed by phosphor image-TLC of enzymatic reaction mixtures as described in “Materials and Methods.” ○, No microsomes added; +, positive control using microsomes prepared from 2-d-old sorghum seedlings; CHO, p-hydroxybenzaldehyde (degradation product of p-hydroxymandelonitrile); OX, Z-p-hydroxyphenylacetaldehyde oxime; and TYR, l-Tyr (which stays at the origin of the TLC plate). B, The cyanide potential of leafs and stems measured after growth in the absence and presence of KNO₃ as described in “Materials and Methods.” In all experiments shown, the induction period used was 12 d.

Figure 7

Nitrate induction of CYP79A1 mRNA in 35-d-old plants. The plants were grown for 35 d in soil where after they were watered with 25 mm KNO₃ or 25 mm KCl for 2 or 5 d. Total RNA was extracted from leaf and stems and used for reverse transcription PCR with CYP79A1 and ac1 actin-specific primers. The primers were placed over introns to exclude amplification from contaminating genomic DNA. Equal amounts of each cDNA was used for PCR.

Figure 8

The effect of nitrate on CYP79A1 and CYP71E1 mRNA levels in sorghum seedlings of different ages. Seeds were imbibed and germinated in water for the time periods indicated and then incubated in 25 mm KCl or 25 mm KNO₃ for 24 h. Total RNA was isolated and analyzed by northern blotting. A, Hybridization with a probe for CYP79A1. B, Hybridization with a probe for CYP71E1. C, Ethidium bromide staining of the RNA (only ribosomal RNA is visible) to confirm that the same amount of RNA was applied to each lane.