Diurnal regulation of cyanogenic glucoside biosynthesis and endogenous turnover in cassava

Abstract

Cyanogenic glucosides are present in many plants, including eudicots, monocots, and ferns and function as defence compounds based on their ability to release hydrogen cyanide. In this study, the diurnal rhythm of cyanogenic glucoside content and of transcripts and enzymes involved in their biosynthesis was monitored in cassava plants grown in a glasshouse under natural light conditions. Transcripts of CYP79D1, CYP79D2, CYP71E7/11, and UGT85K5 were at minimal levels around 9 p.m., increased during the night and decreased following onset of early morning light. Transcripts of UGT85K4 and HNL10 showed more subtle variations with a maximum reached in the afternoon. Western blots showed that the protein levels of CYP71E7/11 and UGT85K4/5 decreased during the light period to a near absence around 4 p.m. and then recovered during the dark period. Transcript and protein levels of linamarase were stable throughout the 24-hr cycle. The linamarin content increased during the dark period. In the light period, spikes in the incoming solar radiation were found to result in concomitantly reduced linamarin levels. In silico studies of the promoter regions of the biosynthetic genes revealed a high frequency of light, abiotic stress, and development-related transcription factor binding motifs. The synthesis and endogenous turnover of linamarin are controlled both at the transcript and protein levels. The observed endogenous turnover of linamarin in the light period may offer a source of reduced nitrogen to balance photosynthetic carbon fixation. The rapid decrease in linamarin content following light spikes suggests an additional function of linamarin as a ROS scavenger.

Keywords: Manihot esculenta; UDPG‐dependent glycosyltransferase; biosynthesis; cytochrome P450; enzyme turnover; linamarin; lotaustralin; pathway regulation; transcriptional regulation.

Figure 1

Biosynthesis and hydrolytic bioactivation of the cyanogenic glucosides linamarin (R = methyl) and lotaustralin (R = ethyl) in cassava (M. esculenta)

Figure 2

Number of transcription binding factor motifs related to abiotic and biotic stresses, circadian rhythm, light responses, development as well as nutrient stress present in the promoter region of genes encoding enzymes involved in the biosynthesis and hydrolytic bioactivation of linamarin and lotaustralin in cassava (M. esculenta)

Figure 3

Diurnal variation of linamarin content in the second fully unfolded leaf of 3‐month‐old cassava (M. esculenta) plants measured by LC‐MS and displayed as area under peak per 10 mm leaf disk calibrated against internal standards. The measured light intensities are shown as a line graph and converted to a color scale presented above the plot. Bars indicate SE, n = 10. ANOVA test: p < .001, F > F _crit

Figure 4

Diurnal variation of protein content of CYP71E7/11, UGT85K4/5, linamarase, and α‐hydroxynitrile lyase (HNL) as monitored by Western blot using specific antibodies. β‐Actin was used as reference protein and monitored using a specific antibody and by Ponceau stain. Color scale below the hour scale indicates light intensities

Figure 5

Diurnal variation of the levels of the gene transcripts encoding the enzymes in the biosynthesis and hydrolytic bioactivation of linamarin and lotaustralin in cassava (M. esculenta) measured by real‐time qPCR with β‐Actin and GAPDH as reference genes. Rubisco small subunit (RubSS) is included as control of reaction to light. Color scale above the plots indicates the differences in light intensities measured at the different time points. Bars indicate SE, n = 10