SummaryNearly one-third of the world's population, mostly women and children, suffer from iron malnutrition and its consequences, such as anaemia or impaired mental development. Iron fortification of food is difficult because soluble iron is either unstable or unpalatable, and non-soluble iron is not bioavailable. Genetic engineering of crop plants to increase iron content has therefore emerged as an alternative for iron biofortification. To date, strategies to increase iron content have relied on single genes, with limited success. Our work focuses on rice as a model plant, because it feeds one-half of the world's population, including the majority of the iron-malnourished population. Using the targeted expression of two transgenes, nicotianamine synthase and ferritin, we increased the iron content of rice endosperm by more than six-fold.Analysis of transgenic rice lines confirmed that, in combination, they provide a synergistic effect on iron uptake and storage. Laser ablation-inductively coupled plasmamass spectrometry showed that the iron in the endosperm of the transgenic rice lines accumulated in spots, most probably as a consequence of spatially restricted ferritin accumulation. Agronomic evaluation of the high-iron rice lines did not reveal a yield penalty or significant changes in trait characters, except for a tendency to earlier flowering. Overall, we have demonstrated that rice can be engineered with a small number of genes to achieve iron biofortification at a dietary significant level.
A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin gene enod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5 and 3 regions of enod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40 action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.RNAs encoding only short open reading frames (sORFRNAs) have received considerable attention in recent years because they show a striking diversity in many cell types from various organisms. Several RNAs exhibit a function without being translated into proteins, for example, tRNAs, rRNAs, RNAs in ribozymes, and small nuclear RNAs from spliceosomes (reviewed in reference 8). However, a pentapeptideencoding sORF present in the 23S rRNA from Escherichia coli was recently shown to render this bacterium resistant to a specific antibiotic (40). In eukaryotes, several sORF-RNAs are induced at specific stages of development, suggesting their participation in various differentiation processes (7,14,18,31,38,40,44,46). Eukaryotic cells may use sORF-RNAs for the regulation of several cellular processes, as suggested by a thorough analysis of the yeast genome leading to identification of several new noncoding and sORF-containing RNAs (31). In eukaryotes, sORFs present in mRNAs are likely to be translated, since translation of mRNAs is achieved by a scanning mechanism in a 5Ј-to-3Ј direction. Indeed, there are several examples where translation of upstream sORFs regulates expression of the 3Ј ORF corresponding to the gene product (15,43). At the same time, very little is known about the fate of the encoded oligopeptides in the cell and whether translation of sORFs present in sORF-RNAs is relevant for gene activity. For example, even though a putative protein product of the H19 RNA was detected using immunological approaches (23), the main function of H19 seems to lie in the mRNA molecule rather than in the encoded protein (24).Leguminous plants have the ability to enter into symbiosis with N 2 -fixing bacteria (collectively called rhizobia) to form the root nodule. Development of this symbiotic organ depends on the coordinate expressio...
Genetic engineering can be used to complement traditional breeding methods in crop plant improvement. Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. The prerequisites for genetic engineering are efficient transformation and tissue culture systems that allow selection and regeneration of transgenic plants. Cassava, an integral plant for food security in developing countries, has until now been recalcitrant to transformation approaches. We report here a method for regenerating stably transformed cassava plants after cocultivation with Agrobacterium tumefaciens, which opens cassava for future improvement via biotechnology.
Tissue-specific or regulated expression of transgenes is desirable in order to prevent pleiotropic side effects of putatively harmful transgene products as well as loss of energy resources due to unnecessary accumulation of transgene products. Epidermis-specific expression would be useful for many defense-related genes directed against attack by fungal pathogens that enter the plant body by direct penetration through the epidermis. In an approach to enhance resistance of wheat to the powdery mildew fungus Blumeria graminis f.sp. tritici, a novel epidermis-specific promoter was developed and used for expression of two defense-related genes. A 2.3 kb fragment of the wheat GstA1 promoter in combination with an intron-containing part of the wheat WIR1a gene was found to drive strong and constitutive transient expression in wheat epidermis. This promoter-intron combination was used for overexpression of oxalate oxidase 9f-2.8 and TaPERO peroxidase, two defense-related wheat genes expressed in inner leaf tissues. Expression studies of several transgenic lines by in situ oxalate-oxidase staining, RNA and protein blot analyses, as well as real-time PCR, demonstrated strong and constitutive transgene expression in the shoot epidermis. Transient as well as stable over-expression of the TaPERO peroxidase gene in wheat epidermis under the control of the GstA1i promoter resulted in enhanced resistance against Blumeria graminis f.sp. tritici, whereas oxalate-oxidase overexpression had no effect in either system. The data suggest that the wheat GstA1 promoter in combination with the WIR1a intron is useful for transgenic approaches to fungal disease resistance in cereals.
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