Biosynthesis and chemical transformation of benzoxazinoids in rye during seed germination and the identification of a rye Bx6-like gene.

Benzoxazinoids are secondary metabolites with plant defense properties and possible health-promoting effects in humans. In this study, the transcriptional activity of ScBx genes (ScBx1-ScBx5; ScBx6-like), involved in benzoxazinoid biosynthesis, was analyzed during germination and early seedling development in rye. Our results showed that ScBx genes had highest levels of expression at 24-30 h after germination, followed by a decrease at later stages. For ScBx1-ScBx5 genes expression was higher in shoots compared with root tissues and vice versa for ScBx6-like gene transcripts. Moreover, methylated forms of benzoxazinoids accumulated in roots rather than in shoots during seedling development, in particular reaching high levels of HMBOA-glc in roots. Chemical profiles of benzoxazinoid accumulation in the developing seedling reflected the combined effects of de novo biosynthesis of the compounds as well as the turnover of compounds either pre-stored in the embryo or de novo biosynthesized. Bioinformatic analysis, together with the differential distribution of ScBx6-like transcripts in root and shoot tissues, suggested the presence of a ZmBx6 homolog encoding a 2-oxoglutarate dependent dehydrogenase in rye. The ScBx6-like cDNA was expressed in E. coli for functional characterization in vitro. LC-MS/MS analysis showed that the purified enzyme was responsible for the oxidation of DIBOA-glc into TRIBOA-glc, strongly suggesting the ScBX6-like enzyme in rye to be a functional ortholog of maize ZmBX6.

Benzoxazinoids: Cereal phytochemicals with putative therapeutic and health-protecting properties.

Benzoxazinoids (BXs) are a group of natural chemical compounds with putative pharmacological and health-protecting properties. BXs were formerly identified in and isolated from selected dicot medicinal plants and young cereal plants. Recently, BXs were found to be present in mature cereal grains and bakery products, such that knowledge about the pharmacological properties of BXs, which until now have unknowingly been consumed through the daily bread and breakfast cereals, has come into new focus. This review discusses published results from in vitro studies and a few human and animal model studies on the health effects and pharmacological responses of various BX compounds. Many of these studies have reported antimicrobial, anticancer, reproductive system stimulatory, central nervous system stimulatory, immunoregulatory, and appetite- and weight-reducing effects of BXs and/or BX derivatives. The health benefits of wholegrain intake may be associated with the solitary and/or overlapping biological effects of fibers, lignans, phenolic acids, alkylresorcinols, BXs, and other bioactive compounds. In the context of BXs as dietary ingredients, further comprehensive investigations are required to understand their biological functions, to elucidate the underlying mechanisms, to explore their potential contribution on the health effects associated with wholegrain consumption, and to examine their potential as functional food ingredients.

Comparison of the levels of bioactive benzoxazinoids in different wheat and rye fractions and the transformation of these compounds in homemade foods.

Benzoxazinoids are important phytochemicals found in wheat and rye that are associated with plant resistance against pathogens, and recent studies have emphasized the potential health-promoting role of these compounds i.e. anti-cancer, anti-allergy and anti-inflammation. Accordingly, an understanding of their distribution in seeds and the effect of different processing techniques on their transformation will be helpful in identifying the mechanisms of their production and distribution and will support the on-going efforts to utilize these compounds in health-promoting food products. The analysis of seed fractions obtained from the milling of wheat and rye showed significantly higher concentrations of these bioactive compounds in the germ than in the other fractions, i.e. the bran and endosperm. Di-hexoses of 2,4-dihydroxy-1, 4-benzoxazin-3-one (DIBOA-glc-hexose) and 2-hydroxy-1, 4-benzoxazin-3-one (HBOA-glc-hexose) were the predominant compounds found in the different wheat and rye seed fractions followed by DIBOA-glc and DIBOA. The soaking and boiling of three rye-based breakfast cereals resulted in considerable changes in the benzoxazinoid contents. The soaking of pearled rye promoted the conversion of DIBOA-glc-hexose into DIBOA-glc. When these cereals were boiled, the increase in the DIBOA-glc content was much lower than that observed for soaking. For rye flakes, the pattern of these benzoxazinoids was different from that in pearled rye seeds. A considerable amount of the benzoxazinoids was also leached into the water during soaking or boiling. This study contributes to the understanding of the underlying processes involved in the biochemical changes of benzoxazinoids and will be the basis for future studies on other food-processing techniques.

Expression dynamics and ultrastructural localization of epitope-tagged Abutilon mosaic virus nuclear shuttle and movement proteins in Nicotiana benthamiana cells.

The geminivirus Abutilon mosaic virus (AbMV) encodes two proteins which are essential for viral spread within plants. The nuclear shuttle protein (NSP) transfers viral DNA between the nucleus and cytoplasm, whereas the movement protein (MP) facilitates transport between cells through plasmodesmata and long-distance via phloem. An inducible overexpression system for epitope-tagged NSP and MP in plants yielded unprecedented amounts of both proteins. Western blots revealed extensive posttranslational modification and truncation for MP, but not for NSP. Ultrastructural examination of Nicotiana benthamiana tissues showed characteristic nucleopathic alterations, including fibrillar rings, when epitope-tagged NSP and MP were simultaneously expressed in leaves locally infected with an AbMV DNA A in which the coat protein gene was replaced by a green fluorescent protein encoding gene. Immunogold labelling localized NSP in the nucleoplasm and in the fibrillar rings. MP appeared at the cell periphery, probably the plasma membrane, and plasmodesmata.

Three C-terminal phosphorylation sites in the Abutilon mosaic virus movement protein affect symptom development and viral DNA accumulation.

The Abutilon mosaic virus (AbMV, Geminiviridae) DNA B component encodes a movement protein (MP), which facilitates viral transport within plants and affects pathogenicity. The presence of phosphorylated serine and threonine residues was confirmed for MP expressed in yeast and Nicotiana benthamiana by comparative Western blot analysis using phospho-amino acid- and MP-specific immunodetection. Mass spectrometry of yeast-derived MP identified three phosphorylation sites located in the C-terminal domain (Thr-221, Ser-223 and Ser-250). To assess their functional relevance in plants, several point mutations were generated in the MP gene of DNA B, which replace Thr-221, Ser-223 and Ser-250, either singly or in combinations, with either an uncharged alanine or a phosphorylation-mimicking aspartate residue. When co-inoculated with DNA A, all mutants were infectious. In systemically infected plants the symptoms and/or viral DNA accumulation were significantly altered for several of the mutants.