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

Abstract

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.

Metal Selectivity Determinants in a Family of Transition Metal Transporters

“…Recent transcriptomics data suggest the presence of metal tolerance proteins (MTPs) 2 that might be responsible for transition metal transport within barley grain (9). MTPs are plant members of the large cation diffusion facilitator (CDF) family of transporters ubiquitous to all forms of life (archaea, bacteria, and eukaryotes) (10).…”

Moving micronutrients from the soil to the seeds: Genes and physiological processes from a biofortification perspective

“…For mineral micronutrients, the transporters that are involved in phloem unloading and uptake by the filial tissues are not well defined, but likely belong to the families discussed as transporters in other parts of the plant. Indeed, laser capture microdissection of barley grain into transfer cells of the maternal vascular bundle, aluerone, endosperm, and embryo fractions, followed by microarray analysis, revealed expression of genes from the following families: HMA, ZIP, MTP, Nramp, NAS, and YSL [166]. Typically the expression was not evenly distributed among the different cell types, suggesting that, as in other tissues, certain transporters are important in specific cell types to move micronutrients through the plant.…”

Barley Metallothioneins: MT3 and MT4 Are Localized in the Grain Aleurone Layer and Show Differential Zinc Binding

“…Samples were run in four biological replicates with two technical replicates each. Quantifications of MT gene expression were done using HvGAPDH as reference (Tauris et al, 2009). As positive controls LTP2 (TC258870), ITR1 (TC139046), and HOR2 (TC138699) were used (Kalla et al, 1994;Diaz et al, 2005).…”