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.

The Understanding of the Plant Iron Deficiency Responses in Strategy I Plants and the Role of Ethylene in This Process by Omic Approaches

“…So far, only one study on proteome profiling in Fe-deficient rice roots and shoots (Chen et al, 2015) and two in maize, with one in Fe-deficient root hairs (Li et al, 2015) and the other in roots (Hopff et al, 2013), have been carried out to investigate the global protein changes or the alterations in the plasma membrane proteome, by means of two-dimensional electrophoresis (2-DE), or 1-DE coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) or LC-MS/MS. By contrast, most of the proteomic studies upon Fe deficiency have been performed in Strategy I species, including Beta vulgaris (Andaluz et al, 2006; Rellan-Alvarez et al, 2010; Gutierrez-Carbonell et al, 2016), Tomato ( Solanum lycopersicum L.) (Brumbarova et al, 2008; Genannt Bonsmann et al, 2008; Muneer and Jeong, 2015), cucumber ( Cucumis sativus ) (Donnini et al, 2010; Li and Schmidt, 2010; Vigani et al, 2017), pea ( Pisum sativum L.) (Meisrimler et al, 2011, 2016), Prunus hybrid GF 677 rootstock ( P. dulcis × P. persica ) (Rodriguez-Celma et al, 2013a), Lupinus texensis (Lattanzio et al, 2013), Hyoscyamus albus (Khandakar et al, 2013), Medicago truncatula (Rodriguez-Celma et al, 2011, 2016), citrus rootstocks (Muccilli et al, 2013), Brassica napu (Gutierrez-Carbonell et al, 2015), Populus cathayana (Zhang S. et al, 2016), and Arabidopsis (Laganowsky et al, 2009; Lan et al, 2011, 2012b; Mai et al, 2015; Pan et al, 2015; Zargar et al, 2015a,b). Most of the protein profiling studies were focused on the global protein changes in the whole roots/shoots, and few of them investigated proteome of the specific plant parts such as root hairs (Li et al, 2015), cellular compartments including root plasma membrane (Hopff et al, 2013), thylakoid membranes (Andaluz et al, 2006), and shoot microsomal fragments (Zargar et al, 2015b), as well as phloem saps (Lattanzio et al, 2013; Gutierrez-Carbonell et al, 2015).…”

Extraction and fractionation of subproteome from root tips of Hyoscyamus albus

“…For sequential sub-proteome fractionation, two IEF solutions were used: IEF solution A (7 M urea, 2 M thiourea, 4% w/v CHAPS, 0.2% carrier ampholytes, 0.002% bromophenol blue dye and 50 mM DTT) and IEF solution B (6 M urea, 2 M thiourea, 2% w/v SB3-10, 1% w/v ASB-14, 0.2% carrier ampholytes, 0.002% bromophenol blue dye, and 100 mM DTT). 2DE was performed as described previously (Khandakar et al, 2013), using Mini-Protean TGX precast gels (AnykD IPG/Prep, Bio-Rad, Hercules, CA, USA). Gels were stained with CBB R-250 (Bio-Rad, Hercules, CA, USA) or with Flamingo fluorescent stain (Bio-Rad, Hercules, CA, USA).…”

Effects of elevated carbon dioxide on contraction force and proteome composition of sea urchin tube feet

“…To accomplish extraction, separation and identification of proteins from a small amount of tube feet, we applied a small-scale proteomic approach (Yamaguchi, 2011;Khandakar et al, 2013) One milliliter of Trizol reagent (Life Technologies, Carlsbad, CA, USA), 100 µg of zirconia beads (0.6 mm in diameter, BMS, Tokyo, Japan), and a stainless-steel bead (5 mm in diameter, BMS, Tokyo, Japan) were added to the frozen tube feet in a 2 mL screw-cap centrifuge tube (Sarstedt, Nümbrecht, Germany). The tubes were mounted in a Master Rack aluminium block (BMS, Tokyo, Japan) and agitated for 2 min at 25ºC in a ShakeMaster Auto ver 1.5 (BMS, Tokyo, Japan).…”