Dhurrin metabolism in the developing grain of Sorghum bicolor (L.) Moench investigated by metabolite profiling and novel clustering analyses of time-resolved transcriptomic data

Abstract

BackgroundThe important cereal crop Sorghum bicolor (L.) Moench biosynthesize and accumulate the defensive compound dhurrin during development. Previous work has suggested multiple roles for the compound including a function as nitrogen storage/buffer. Crucial for this function is the endogenous turnover of dhurrin for which putative pathways have been suggested but not confirmed.ResultsIn this study, the biosynthesis and endogenous turnover of dhurrin in the developing sorghum grain was studied by metabolite profiling and time-resolved transcriptome analyses. Dhurrin was found to accumulate in the early phase of grain development reaching maximum amounts 25 days after pollination. During the subsequent maturation period, the dhurrin content was turned over, resulting in only negligible residual dhurrin amounts in the mature grain. Dhurrin accumulation correlated with the transcript abundance of the three genes involved in biosynthesis. Despite the accumulation of dhurrin, the grains were acyanogenic as demonstrated by the lack of hydrogen cyanide release from macerated grain tissue and by the absence of transcripts encoding dhurrinases. With the missing activity of dhurrinases, the decrease in dhurrin content in the course of grain maturation represents the operation of hitherto uncharacterized endogenous dhurrin turnover pathways. Evidence for the operation of two such pathways was obtained by metabolite profiling and time-resolved transcriptome analysis. By combining cluster- and phylogenetic analyses with the metabolite profiling, potential gene candidates of glutathione S-transferases, nitrilases and glycosyl transferases involved in these pathways were identified. The absence of dhurrin in the mature grain was replaced by a high content of proanthocyanidins. Cluster- and phylogenetic analyses coupled with metabolite profiling, identified gene candidates involved in proanthocyanidin biosynthesis in sorghum.ConclusionsThe results presented in this article reveal the existence of two endogenous dhurrin turnover pathways in sorghum, identify genes putatively involved in these transformations and show that dhurrin in addition to its insect deterrent properties may serve as a storage form of reduced nitrogen. In the course of sorghum grain maturation, proanthocyanidins replace dhurrin as a defense compound. The lack of cyanogenesis in the developing sorghum grain renders this a unique experimental system to study CNglc synthesis as well as endogenous turnover.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3360-4) contains supplementary material, which is available to authorized users.

Counting the costs: nitrogen partitioning in Sorghum mutants

“…This could potentially mobilise additional nitrogen resources leading to improved growth, particularly at low nitrogen treatments, and is consistent with our observations. This is supported by the results from the analysis of the developing sorghum grain, which showed that although the mature grain is acyanogenic, dhurrin levels are quite high in the immature grains (Nielsen et al 2016). Concomitant with this decrease in dhurrin is an increase in NIT4A and NIT4B2 expression, indicating that the endogenous turnover pathway operates in the seed to provide an alternative nitrogen source for seed storage protein production (Nielsen et al 2016).…”

“…Dhurrin synthesis has been shown to be transcriptionally regulated in seedlings (Busk and Møller 2002) and the developing grain (Nielsen et al 2016). Here, CYP79A1 transcript levels were very low in the leaf and root tissue of plants at the three-and five-leaf stage, even when HCNp was quite high.…”

“…The biosynthetic gene CYP79A1 encodes the enzyme catalysing the first committed step in dhurrin biosynthesis. At some stages of sorghum ontogeny, dhurrin synthesis is under transcriptional control (Busk and Møller 2002;Nielsen et al 2016). Jenrich et al (2007) showed that the nitrilases, SbNIT4A, SbNIT4B1 and SbNIT4B2, form heteroduplexes that are involved in the detoxification of HCN (SbNIT4A/B1 and/or SbNIT4A/4B2), whilst only SbNIT4A/4B2 forms an active complex in the alternative nitrogen turnover pathway ( Fig.…”

Abiotic Stress Effects on Sorghum Leaf Dhurrin and Soluble Sugar Contents throughout Plant Development

“…This is evidenced by the increase in significance of the dhurrin–glucose relationships ( r = 0.41 in the control to r = 0.61 in the preflowering treatment block) under preflowering water deficit. It has been proven that under environmental variations, dhurrin can be catabolized to release HCN through the cyanogenesis pathway (Morant et al, 2008) or turned over, thus releasing glucose and ammonia as byproducts through the alternate putative pathway (Adewusi, 1990; Piotrowski et al, 2001; Busk and Møller, 2002; Nielsen et al, 2016). As stress prolongs, the relationships between dhurrin and the sugars decrease in significance, suggesting a complete slowdown of leaf metabolic processes as the plant becomes less reliant on the leaves and more on the remobilization of its stored stem assimilates for its developmental processes.…”

Cyanogenesis in Macadamia and Direct Analysis of Hydrogen Cyanide in Macadamia Flowers, Leaves, Husks, and Nuts Using Selected Ion Flow Tube–Mass Spectrometry

“…Most characterization studies in Macadamia and other plants only involved analysis of cyanogenic glycosides using time-consuming assays coupled with HPLC analysis [6,25,26]. Furthermore, typical analysis of releasable cyanide uses tedious assays and subsequent spectrophotometry or LC-or GC-MS analysis [14,27,28]. In this study, hydrogen cyanide was measured directly above the headspace of the different parts of the Macadamia plant including the flowers, leaves, husks, and nuts using selected ion flow tube-mass spectrometry (SIFT-MS).…”