A small-scale proteomic approach reveals a survival strategy, including a reduction in alkaloid biosynthesis, in Hyoscyamus albus roots subjected to iron deficiency

Abstract

Hyoscyamus albus is a well-known source of the tropane alkaloids, hyoscyamine and scopolamine, which are biosynthesized in the roots. To assess the major biochemical adaptations that occur in the roots of this plant in response to iron deficiency, we used a small-scale proteomic approach in which 100 mg of root tips were treated with and without Fe, respectively, for 5 days. Two-dimensional mini gels showed that 48 spots were differentially accumulated between the two conditions of Fe availability and a further 36 proteins were identified from these spots using MALDI-QIT-TOF mass spectrometry. The proteins that showed elevated levels in the roots lacking Fe were found to be associated variously with carbohydrate metabolism, cell differentiation, secondary metabolism, and oxidative defense. Most of the proteins involved in carbohydrate metabolism were increased in abundance, but mitochondrial NAD-dependent malate dehydrogenase was decreased, possibly resulting in malate secretion. Otherwise, all the proteins showing diminished levels in the roots were identified as either Fe-containing or ATP-requiring. For example, a significant decrease was observed in the levels of hyoscyamine 6β-hydroxylase (H6H), which requires Fe and is involved in the conversion of hyoscyamine to scopolamine. To investigate the effects of Fe deficiency on alkaloid biosynthesis, gene expression studies were undertaken both for H6H and for another Fe-dependent protein, Cyp80F1, which is involved in the final stage of hyoscyamine biosynthesis. In addition, tropane alkaloid contents were determined. Reduced gene expression was observed in the case of both of these proteins and was accompanied by a decrease in the content of both hyoscyamine and scopolamine. Finally, we have discussed energetic and Fe-conservation strategies that might be adopted by the roots of H. albus to maintain iron homeostasis under Fe-limiting conditions.

Keywords: Fe deficiency; Hyoscyamus albus; hyoscyamine 6β-hydroxylase; malic acid secretion; roots; small-scale proteomics; tropane alkaloid biosynthesis.

Figure 1

2-D Gel electrophoretic separations of Hyoscyamus albus root-tip proteins extracted from cultures exposed to Fe-replete (A) and Fe-deficient (B) conditions. Twenty μg of proteins extracted from 100 mg of H. albus root tips cultured under Fe-replete (A) and Fe-deficient (B) conditions for 5 days were separated on 7 cm IPG strips (pH5-8 linear gradient) using isoelectric focusing (IEF) in the first dimension, followed by AnyKd® gels in the second dimension. Gels were stained with Flamingo fluorescent stain. The prodigy rank number was used as a spot number; the number shown in yellow color is identified as hyoscyamine 6β-hydroxylase (H6H), the others are shown in black color. Molecular mass (kDa) and pI are indicated on the left-hand and upper axes, respectively.

Figure 2

Classification of differentially accumulated proteins from the root tips of H. albus roots cultured under Fe-deficient and Fe-replete conditions. A total of 36 identified proteins were found to be differentially accumulated and were classified into six functional categories, viz: carbohydrate metabolism; defense response; amino acid/protein metabolism; structure/development; electron transport chains (ETC)/ATP synthesis; and secondary metabolism/others. Blue and red bars indicate up and down proteins in abundance, respectively.

Figure 3

Citric and malic acids released into the culture medium by H. albus roots under Fe-deficient conditions. After H. albus root cultures had been exposed to Fe-deficient culture medium, 500 μL of the medium was collected at days 1, 2, 3, and 5, respectively. Results are the means of three independent experiments, and bars indicate standard deviations of the means.

Figure 4

Transcript accumulations of H6H, Cyp80F1 and RibD in the root tips of H. albus roots cultured under Fe-deficient and Fe-replete conditions. Representative expression profiles are shown in (A) and (C) and semiquantitative analysis of the expressions with three biological replicates and standard deviations are in (B) and (D), respectively. H6H, hyoscyamine 6β-hydoroxylase, catalyzes the conversion from hyoscyamine to scopolamine, Cyp80F1, a cytochrome P450, is involved in littorine rearrangement to produce hyoscyamine and RibD, riboflavin synthase, is involved in de nono synthesis of ribofalvin. RNA was extracted from 0.1 g of fresh root tips, and 500–1000 ng RNA was used for RT-PCR. Using primers specific for H6H, Cyp80F1 and RibD, corresponding fragments of 512 bp, 202 bp and 215 bp, respectively, were amplified. Ribosomal RNA 18S universal primer pairs were used as an internal standard for normalization (product fragment, 315 bp). −Fe, no addition of Fe; +Fe, with addition of 0.1 mM Fe(III)-EDTA.

Figure 5

Alkaloid contents of the tips and of the remaining older parts of H. albus roots cultured under Fe deficiency and Fe repletion. Roots were treated with and without Fe for 5 days, and then harvested. Roots were separated into the tips (ca. 1.0–1.5 cm in length) and the remaining parts, and then dried at 50°C overnight. Results are means of three independent experiments, and bars indicate standard deviations of the means.