Targeted expression of HvHMA2 increases the mineral content of the inner endosperm in barley.

Cereals are a major source of dietary energy and protein but are nutritionally poor in micronutrients. Zinc (Zn) biofortification of staple crops has been proposed as a promising strategy to combat the global challenge of human Zn-deficiency. The aim of this study was to improve the Zn content in the edible part of the barley (Hordeum vulgare L.) grain by enhancing Zn translocation into the developing seeds. We demonstrate that the barley plasma membrane P-type ATPase Zn transporter, HvHMA2 is an efficient candidate for mineral biofortification of crops. Following a cisgenic approach to produce transgenic homozygous barley line over-expressing HvHMA2 in the transfer cells of the grain, resulted in a doubling of a wide range of nutrients including Zn, iron (Fe), and magnesium (Mg) in the inner endosperm. This article is protected by copyright. All rights reserved.

A roadmap for zinc trafficking in the developing barley grain based on laser capture microdissection and gene expression profiling.

Nutrients destined for the developing cereal grain encounter several restricting barriers on their path towards their final storage sites in the grain. In order to identify transporters and chelating agents that may be involved in transport and deposition of zinc in the barley grain, expression profiles have been generated of four different tissue types: the transfer cells, the aleurone layer, the endosperm, and the embryo. Cells from these tissues were isolated with the 'laser capture microdissection' technology and the extracted RNA was subjected to three rounds of T7-based amplification. The amplified RNA was subsequently hybridized to Affymetrix 22K Barley GeneChips. Due to the short average length of the amplified transcripts and the positioning of numerous probe sets at locations more than 400 base pairs (bp) from the poly(A)-tail, a normalization approach was used where the probe positions were taken into account. On the basis of the expression levels of a number of metal homeostasis genes, a working model is proposed for the translocation of zinc from the phloem to the storage sites in the developing grain.

UCE: A uracil excision (USER)-based toolbox for transformation of cereals.

BACKGROUND: Cloning of gene casettes and other DNA sequences into the conventional vectors for biolistic or Agrobacterium-mediated transformation is hampered by a limited amount of unique restriction sites and by the difficulties often encountered when ligating small single strand DNA overhangs. These problems are obviated by "The Uracil Specific Excision Reagent (USER)" technology (New England Biolabs) which thus offers a new and very time-efficient method for engineering of big and complex plasmids. RESULTS: By application of the USER system, we engineered a collection of binary vectors, termed UCE (USER cereal), ready for use in cloning of complex constructs into the T-DNA. A series of the vectors were tested and shown to perform successfully in Agrobacterium-mediated transformation of barley (Hordeum vulgare L.) as well as in biolistic transformation of endosperm cells conferring transient expression. CONCLUSIONS: The USER technology is very well suited for generating a toolbox of vectors for transformation and it opens an opportunity to engineer complex vectors, where several genetic elements of different origin are combined in a single cloning reaction.