Sergei Storozhenko

A Mutation of the Mitochondrial ABC Transporter Sta1 Leads to Dwarfism and Chlorosis in the Arabidopsis Mutant starik

A mutation in the Arabidopsis gene STARIK leads to dwarfism and chlorosis of plants with an altered morphology of leaf and cell nuclei. We show that the STARIK gene encodes the mitochondrial ABC transporter Sta1 that belongs to a subfamily of Arabidopsis half-ABC transporters. The severity of the starik phenotype is suppressed by the ectopic expression of the STA2 homolog; thus, Sta1 function is partially redundant. Sta1 supports the maturation of cytosolic Fe/S protein in Δatm1 yeast, substituting for the ABC transporter Atm1p. Similar to Atm1p-deficient yeast, mitochondria of the starik mutant accumulated more nonheme, nonprotein iron than did wild-type organelles. We further show that plant mitochondria contain a putative l-cysteine desulfurase. Taken together, our results suggest that plant mitochondria possess an evolutionarily conserved Fe/S cluster biosynthesis pathway, which is linked to the intracellular iron homeostasis by the function of Atm1p-like ABC transporters.

Folate fortification of rice by metabolic engineering

Rice, the worlds major staple crop, is a poor source of essential micronutrients, including folates (vitamin B9). We report folate biofortification of rice seeds achieved by overexpressing two Arabidopsis thaliana genes of the pterin and para-aminobenzoate branches of the folate biosynthetic pathway from a single locus. We obtained a maximal enhancement as high as 100 times above wild type, with 100 g of polished raw grains containing up to four times the adult daily folate requirement.

Folates and Folic Acid: From Fundamental Research Toward Sustainable Health

Folates are of paramount importance in one-carbon metabolism of most organisms. Plants and microorganisms are able to synthesize folates de novo, making them the main dietary source for humans and animals, which are dependent on food or feed supplies for folates. Folate deficiency is an increasing problem in the developing, as well as in the developed regions of the world, affecting millions of people. Different strategies, such as food fortification and folic acid supplementation, remain far from accessible for the poor rural populations in developing countries. Increasing knowledge concerning folate biosynthesis, transport and catabolism does not only deepen our insight on the regulation of folate metabolism but also provides the keys towards folate enhancement through metabolic engineering in bacteria, as well as in plants. Recently, promising results were obtained using such an approach, but further fundamental research is a prerequisite to develop a practicable solution to fight folate deficiency. In parallel, progress in the development and improvement of folate analysis has been made. Here, we provide the state-of-the-art of folate biosynthesis, catabolism, and salvage. Finally, we report on progress in folate biofortification and discuss the agroeconomical aspect of biofortified crop plants.

γ-Glutamyl Transpeptidase in Transgenic Tobacco Plants. Cellular Localization, Processing, and Biochemical Properties

γ-Glutamyl transpeptidase (γ-GT) is a ubiquitous enzyme that catalyzes the first step of glutathione (GSH) degradation in the γ-glutamyl cycle in mammals. A cDNA encoding an Arabidopsis homolog for γ-GT was overexpressed in tobacco (Nicotiana tabacum) plants. A high level of the membrane-bound γ-GT activity was localized outside the cell in transgenic plants. The overproduced enzyme was characterized by a high affinity to GSH and was cleaved post-translationally in two unequal subunits. Thus, Arabidopsis γ-GT is similar to the mammalian enzymes in enzymatic properties, post-translational processing, and cellular localization, suggesting analogous biological functions as a key enzyme in the catabolism of GSH.

Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for the sensitive determination of folates in rice

High-performance liquid chromatography, coupled to tandem mass spectrometry (HPLC-MS/MS) has been established as the method of choice for the sensitive and simultaneous determination of different folates in a particular matrix, especially when only minute quantities of material are available. Using a previously developed and validated HPLC-MS/MS method as a starting point, we here report on the development and validation of an ultra-performance liquid chromatography (UPLC-MS/MS) method for analysis of folates in rice, which allows higher throughput and better resolution. UPLC was performed under gradient conditions on an Acquity HSS T3 column, followed by tandem mass spectrometry detection. The method was validated based on linearity, sensitivity, precision, accuracy and matrix effects. The limits of detection and the lower limits of quantification varied between 0.06 and 0.45 microg/100 g and 0.12 and 0.91 microg/100 g, respectively. Two linear calibration curves were established, one for the low and the other for the high concentration range. Analysis of the distribution and levels of folates in wild-type and folate-biofortified rice showed up to 50-fold enrichment in biofortified rice, with total folate levels of up to 900 microg/100 g rice. This is the first successful implementation of a UPLC method for the rapid and sensitive quantitative determination of folates in plant material.

Optimisation and validation of a liquid chromatography–tandem mass spectrometry method for folates in rice

An LC-MS/MS method has been developed and validated for the analysis of six monoglutamate folates in rice. Accurate determination of folates in rice seeds required the optimisation of an extraction procedure encompassing homogenisation, heat treatment, tri-enzyme treatment (alpha-amylase, protease and deconjugase), centrifugation and ultrafiltration. The resulting extract was loaded onto a Polaris C 18-A column, followed by gradient elution and detection by tandem mass spectrometry. The validated method is sensitive, reproducible, accurate, and linear between 1 and 400 microg/100g rice (dry weight). The limit of detection and limit of quantification in diluted rice matrix ranged between 0.2 and 1.2 microg/100g and between 0.6 and 4 microg/100g, respectively, depending on the compound. Intra- and inter-day precision varied, respectively, from 1.0 to 9.1% RSD, and from 3.4 to 13.5% RSD, except for one compound (19.1% at low concentrations). The accuracy ranged from 96.9 to 115.4%. Matrix effects were compensated for by use of isotopically labelled internal standards. Most folates in rice extracts were stable when kept for 2 weeks at -20 and -80 degrees C, but degraded after three freeze-thaw cycles or at 4 degrees C, even when stored protected from light. Our validated method offers a sensitive and versatile approach to determine the level of six different monoglutamate folates in a complex matrix as rice.

Identification of an Arabidopsis thaliana cDNA encoding a HSP70‐related protein belonging to the HSP110/SSE1 subfamily

Heat‐shock protein 70 (HSP70)‐related proteins are classified in two main subfamilies: the DnaK subfamily and the HSP110/SSE1 subfamily. We have characterized the first plant member of the HSP110/SSE1 subfamily, HSP91. At least two, tightly linked genes encoding HSP91 are present per haploid Arabidopsis genome. HSP91 is constitutively expressed in non‐stressed Arabidopsis plants and is transiently induced by heat shock.

Health impact in China of folate-biofortified rice.

volume 28 number 6 June 2010 nature biotechnology adverse health outcomes, such as NTDs. In the following analysis, we present the first attempt to evaluate the health impact of folate-biofortified rice. Analogous to previous impact studies of other biofortified staple crops5, we apply the disability-adjusted life years (DALYs) approach6 to evaluate the potential health benefits of rice with a high folate content in China. Several interventions are available to increase folate intake levels in malnourished populations, including folic acid pills supplementation, folic acid fortification and dietary diversification to increase the consumption of folate-rich foods. Implementation of these approaches can often be problematic, however. For example, in poor, rural regions, such as Shanxi Province in Northern China or Balrampur District in Northern India, where industrial fortification is not well established, folate pill distribution often does not reach the targeted individuals and dietary habits are difficult to alter. In this context, folate biofortification (that is, improving the folate content of staple crops) offers an additional approach for alleviating the burden of folate deficiency (Supplementary Discussion, section 5)2. China is an important study location to evaluate folate-biofortified rice for two major reasons. First, it is not only the world leader in the production and consumption of this staple crop, but also considered one of the pioneers of R&D and commercialization of genetically modified (GM) rice7. In 2009, for instance, China’s Ministry of Agriculture issued a bio-safety certificate to pest-resistant Bacillus thuringiensis toxin (Bt) rice, which should lead to large-scale production of this transgenic crop in about 2 to 3 years8. This makes rice, the world’s main staple crop, an appropriate food vehicle for folate biofortification. Second, China as a riceconsuming country is characterized by large folate deficiencies and high NTD prevalence rates9. Each year, about 18,000 pregnancies in China are affected with an NTD (Supplementary Discussion, Table 4). Because of significant differences in rice consumption, folate status and the prevalence of NTDs between the northern and southern regions, a regional comparison of the health impact of folate biofortification in China will further underpin its ex-ante evaluation. Shanxi Province for instance, has one of the highest reported NTD prevalence rates in the world10, in part because folate intake with and recover from drought. But we hope that our model can inform the research and development process of looming downstream challenges for drought-tolerant varieties. For example, our modeling exercise emphasizes not only the importance of generalized gains in water use efficiency and early maturation traits that confer benefits across a broader range of rainfall outcomes, but also the importance of pricing; indeed, diffusion of drought-tolerant crops is likely to be especially sluggish among vulnerable farmers if their seeds cost more than conventional crops. If we seek to ensure the efficient uptake of drought-resistant varieties, demonstrating effectiveness in laboratories and test plots will be only part of a solution. The quandary of a marginal farmer in drought-prone Africa trying to figure out whether his neighbor’s maize really did better than his own emphasizes how adoption of such varieties is unlikely to be as smooth and rapid as experienced previously with Bt cotton.

Improving folate (vitamin B9) stability in biofortified rice through metabolic engineering.

Biofortification of staple crops could help to alleviate micronutrient deficiencies in humans. We show that folates in stored rice grains are unstable, which reduces the potential benefits of folate biofortification. We obtain folate concentrations that are up to 150 fold higher than those of wild-type rice by complexing folate to folate-binding proteins to improve folate stability, thereby enabling long-term storage of biofortified high-folate rice grains.

Cytosolic Hydroxymethyldihydropterin Pyrophosphokinase/Dihydropteroate Synthase from Arabidopsis thaliana A SPECIFIC ROLE IN EARLY DEVELOPMENT AND STRESS RESPONSE

In plants, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase (mitHPPK/DHPS) is a bifunctional mitochondrial enzyme, which catalyzes the first two consecutive steps of tetrahydrofolate biosynthesis. Mining the Arabidopsis genome data base has revealed a second gene encoding a protein that lacks a potential transit peptide, suggesting a cytosolic localization of the isoenzyme (cytHPPK/DHPS). When the N-terminal part of the cytHPPK/DHPS was fused to green fluorescent protein, the fusion protein appeared only in the cytosol, confirming the above prediction. Functionality of cytHPPK/DHPS was addressed by two parallel approaches: first, the cytHPPK/DHPS was able to rescue yeast mutants lacking corresponding activities; second, recombinant cytHPPK/DHPS expressed and purified from Escherichia coli displayed both HPPK and DHPS activities in vitro. In contrast to mitHPPK/DHPS, which was ubiquitously expressed, the cytHPPK/DHPS gene was exclusively expressed in reproductive tissue, more precisely in developing seeds as revealed by histochemical analysis of a transgenic cytHPPK/DHPS promoter-GUS line. In addition, it was observed that expression of cytHPPK/DHPS mRNA was induced by salt stress, suggesting a potential role of the enzyme in stress response. This was supported by the phenotype of a T-DNA insertion mutant in the cytHPPK/DHPS gene, resulting in lower germination rates as compared with the wild-type upon application of oxidative and osmotic stress.