Søren Borg

Zinc biofortification of cereals: problems and solutions

The goal of biofortification is to develop plants that have an increased content of bioavailable nutrients in their edible parts. Cereals serve as the main staple food for a large proportion of the world population but have the shortcoming, from a nutrition perspective, of being low in zinc and other essential nutrients. Major bottlenecks in plant biofortification appear to be the root-shoot barrier and--in cereals--the process of grain filling. New findings demonstrate that the root-shoot distribution of zinc is controlled mainly by heavy metal transporting P1B-ATPases and the metal tolerance protein (MTP) family. A greater understanding of zinc transport is important to improve crop quality and also to help alleviate accumulation of any toxic metals.

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.

Iron transport, deposition and bioavailability in the wheat and barley grain

In recent years, the increasing knowledge on the molecular mechanisms underlying mineral uptake, transport, homeostasis and deposition within plants, has paved the way for a more targeted approach to improving the nutrient status of crop plants based on biotechnology. In the present paper we will briefly review existing knowledge on the distribution and transport pathways of iron in the two small grained cereals, barley and wheat, and focus on the efforts made to increase the iron content in cereals in general. However, mineral content is not the only factor of relevance for improving the nutritional status of poor populations. It is thus well documented that a number of plant components can act either as promoters or inhibitors of mineral uptake in the human digestive system (Frossard et al. J Sci Food Agric 80, 817-879 2000; Brinch-Pedersen et al. J Cereal Sci 46, 308-326 2007). The nutritional impact of increasing mineral content accordingly has to be seen in the context of mineral bioavailability. Finally, we will briefly report on recent data from barley, where laser capture microdissection of the different grain tissues combined with gene expression profiling has provided some insight into metal transport and deposition (Tauris et al. 2009). In the present paper we will provide a tentative and preliminary roadmap for iron trafficking in the barley grain.

Plant cell growth and differentiation may involve GAP regulation of Rac activity.

Two Rac GTPase cDNAs, LjRac1 and LjRac2, were identified in the legume Lotus japonicus. Two‐hybrid screening with dominant‐constitutive mutations in the two Rac GTPases target three plant cDNAs, LjRacGAP1, LjRacGAP2 and LjRacGAP3, that encode putative GTPase activating proteins of Rho‐GTPase subfamily members. Employing Rac antiserum, purified recombinant LjRac GTPases and recombinant LjRacGAP1, for ligand overlay assays, in vitro GAP affinity assays and GTPase activation, we confirmed that eukaryote Rac/RacGAP interplay is conserved in plants. In this investigation we have developed some tools that can be used to characterize the role of enhanced LjRac2 expression in developing root nodules.

Dissecting plant iron homeostasis under short and long-term iron fluctuations

A wealth of information on the different aspects of iron homeostasis in plants has been obtained during the last decade. However, there is no clear road-map integrating the relationships between the various components. The principal aim of the current review is to fill this gap. In this context we discuss the lack of low affinity iron uptake mechanisms in plants, the utilization of a different uptake mechanism by graminaceous plants compared to the others, as well as the roles of riboflavin, ferritin isoforms, nitric oxide, nitrosylation, heme, aconitase, and vacuolar pH. Cross-homeostasis between elements is also considered, with a specific emphasis on the relationship between iron homeostasis and phosphorus and copper deficiencies. As the environment is a crucial parameter for modulating plant responses, we also highlight how diurnal fluctuations govern iron metabolism. Evolutionary aspects of iron homeostasis have so far attracted little attention. Looking into the past can inform us on how long-term oxygen and iron-availability fluctuations have influenced the evolution of iron uptake mechanisms. Finally, we evaluate to what extent this homeostastic road map can be used for the development of novel biofortification strategies in order to alleviate iron deficiency in human.

A Lotus japonicus cDNA encoding an alpha subunit of a heterotrimeric G-protein.

We are studying the molecular genetics of organ development and nitrogen assimilatory processes in legumes, employing Lotus japonicus as a model experimental plant (Handberg and Stougaard, 1992). Inspired by the fact that GTPbinding proteins (GTPases) regulate a multitude of cellular processes, one of our goals is to establish whether GTPases are associated with the aforementioned legume processes. Thus, we are isolating putative GTPase-encoding sequences from DNA libraries of this plant species. The GTP-binding proteins in eukaryotic organisms include factors from the protein translation machinery (Anthony et al., 1990) and the tubulins (Mitchison, 1993) and dynamins (Chen et al., 1991) of the microtubule apparatus. The small GTP-binding proteins include the p21 ras protein that is involved with receptor-mediated cell signaling (Schlessinger, 1993) and the rho proteins that associate with actin cytoskeleton and regulate cell morphology and orientation (Yang and Watson, 1993). The sar, ypt, and rab proteins, associated with endoand exocytosis events, mediate secretion and compartmentalization through vesicle traffic (Novick and Brennwald, 1993). Moreover, a large set of heterotrimeric G-proteins, composed of the guanine nucleotide-binding a subunit and the membrane-associated fi and y subunits, regulate a complex pattern of interactions that are involved with cellular metabolism and signaling (Hepler and Gilman, 1992). In plant cells there is evidence for most of these types of GTP-binding proteins. It has already been noted that there are ypt/rab GTPases involved in endocytosis of the endosymbiont bacteria of legume root nodules (Cheon et al., 1993). With respect to plant heterotrimeric G-proteins, only a few data are available. Useful information about studies of the biological functions, structures, and cytology of plant heterotrimeric G-proteins can be obtained from Weiss et al. (1993) and from references therein. We present here the structural analysis of four L. japonicus cDNA clones, one full length and three partial, obtained by screening of 500,000 recombinant A-plaques from a rootnodule cDNA library. This cDNA analysis yields the complete reading frame of a putative L. japonicus Ga-subunit, LjGPAl

Barley HvHMA1 Is a Heavy Metal Pump Involved in Mobilizing Organellar Zn and Cu and Plays a Role in Metal Loading into Grains

Heavy metal transporters belonging to the P1B-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. Heavy metal transporters belonging to the P1B-ATPase subfamily of P-type ATPases are key players in cellular heavy metal homeostasis. In this study we investigated the properties of HvHMA1, which is a barley orthologue of Arabidopsis thaliana AtHMA1 localized to the chloroplast envelope. HvHMA1 was localized to the periphery of chloroplast of leaves and in intracellular compartments of grain aleurone cells. HvHMA1 expression was significantly higher in grains compared to leaves. In leaves, HvHMA1 expression was moderately induced by Zn deficiency, but reduced by toxic levels of Zn, Cu and Cd. Isolated barley chloroplasts exported Zn and Cu when supplied with Mg-ATP and this transport was inhibited by the AtHMA1 inhibitor thapsigargin. Down-regulation of HvHMA1 by RNA interference did not have an effect on foliar Zn and Cu contents but resulted in a significant increase in grain Zn and Cu content. Heterologous expression of HvHMA1 in heavy metal-sensitive yeast strains increased their sensitivity to Zn, but also to Cu, Co, Cd, Ca, Mn, and Fe. Based on these results, we suggest that HvHMA1 is a broad-specificity exporter of metals from chloroplasts and serve as a scavenging mechanism for mobilizing plastid Zn and Cu when cells become deficient in these elements. In grains, HvHMA1 might be involved in mobilizing Zn and Cu from the aleurone cells during grain filling and germination.

Iron and zinc complexation in wild-type and ferritin-expressing wheat grain: implications for mineral transport into developing grain

We have used synchrotron-based X-ray fluorescence and absorption techniques to establish both metal distribution and complexation in mature wheat grains. In planta, extended X-ray absorption fine structure (EXAFS) spectroscopy reveals iron phytate and zinc phytate structures in aleurone cells and in modified aleurone cells in the transfer region of the grain: iron is coordinated octahedrally by six oxygen atoms and fewer than two phosphorous atoms. Zinc is coordinated tetrahedrally by four oxygen atoms and approximately 1.5 phosphorus atoms in an asymmetric coordination shell. We also present evidence of modified complexation of both metals in transgenic grain overexpressing wheat ferritin. For zinc, there is a consistent doubling of the number of complexing phosphorus atoms. Although there is some EXAFS evidence for iron phytate in ferritin-expressing grain, there is also evidence of a structure lacking phosphorus. This change may lead to an excess of phosphorus within the storage regions of grain, and in turn to the demonstrated increased association of phosphorus with zinc in ferritin-expressing grains. Derivative X-ray absorption spectra also suggest that mineral complexation in the transfer region of ferritin-expressing grains is quite different from that in wild-type grain. This may explain why the raised levels of minerals transported to the developing grain accumulate within the crease region of the transgenic grain.

Molecular analysis of two Ypt/Rab-related sequences isolated from soybean (Glycine max) DNA libraries

From nodule and seedling cDNA libraries we isolated cDNA copies of two mRNAs, derived from the genes gmrl and gmr2, encoding members of the Ypt/Rab family of small GTP-binding proteins. Two deduced protein products, GMR1 and GMR2, were found to be nearly identical differing by only four amino acids in the analysed parts. The two putative proteins are 79% identical to the previously described ARA small GTPase from Arabidopsis thaliana. The GMR proteins may thus be the counterpart of the ARA protein and may perform a related biological function in Glycine max. The gmr2 genomic sequence was isolated and structurally analysed. Expression analyses by northern and cDNA-based PCR showed that the gmr1 and gmr2 genes are constitutively expressed in different plant organs, although at a slightly higher level in callus culture. The classification of the gmr sequences as relatives of the Ypt/Rab family suggests that the deduced GMR proteins are involved in control of processes related to vesicle trafficking in plant cells.

Improving zinc accumulation in cereal endosperm using HvMTP1, a transition metal transporter

Summary Zinc (Zn) is essential for all life forms, including humans. It is estimated that around two billion people are deficient in their Zn intake. Human dietary Zn intake relies heavily on plants, which in many developing countries consists mainly of cereals. The inner part of cereal grain, the endosperm, is the part that is eaten after milling but contains only a quarter of the total grain Zn. Here, we present results demonstrating that endosperm Zn content can be enhanced through expression of a transporter responsible for vacuolar Zn accumulation in cereals. The barley (Hordeum vulgare) vacuolar Zn transporter HvMTP1 was expressed under the control of the endosperm‐specific D‐hordein promoter. Transformed plants exhibited no significant change in growth but had higher total grain Zn concentration, as measured by ICP‐OES, compared to parental controls. Compared with Zn, transformants had smaller increases in concentrations of Cu and Mn but not Fe. Staining grain cross sections with the Zn‐specific stain DTZ revealed a significant enhancement of Zn accumulation in the endosperm of two of three transformed lines, a result confirmed by ICP‐OES in the endosperm of dissected grain. Synchrotron X‐ray fluorescence analysis of longitudinal grain sections demonstrated a redistribution of grain Zn from aleurone to endosperm. We argue that this proof‐of‐principle study provides the basis of a strategy for biofortification of cereal endosperm with Zn.