Felipe Klein Ricachenevsky

Identification of up-regulated genes in flag leaves during rice grain filling and characterization of OsNAC5, a new ABA-dependent transcription factor

Rice is a poor source of micronutrients such as iron and zinc. To help clarify the molecular mechanisms that regulate metal mobilization from leaves to developing seeds, we conducted suppression subtractive hybridization analysis in flag leaves of two rice cultivars. Flag leaves are the major source of remobilized metals for developing seeds. We isolated 78 sequences up-regulated in flag leaves at the grain filling stage relative to the panicle exertion stage. Differential expression of selected genes (encoding 7 transport proteins, the OsNAS3 enzyme and the OsNAC5 transcription factor) was confirmed by quantitative RT-PCR. We show that OsNAC5 expression is up-regulated by natural (aging) and induced senescence processes (dark, ABA application, high salinity, cold and Fe-deficiency) and its expression is not affected in the presence of 6-benzylaminopurine (a senescence inhibitor) under dark-induced senescence. Salt induction of OsNAC5 expression is abolished by nicotinamide, an inhibitor of ABA effects. This result and the presence of cis-acting elements in the promoter region of the OsNAC5 gene suggest an ABA-dependent regulation. Using four different rice cultivars, we show that OsNAC5 up-regulation is higher and earlier in flag leaves and panicles of IR75862 plants, which have higher seed concentrations of Fe, Zn and protein. We suggest that OsNAC5 is a novel senescence-associated ABA-dependent NAC transcription factor and its function could be related to Fe, Zn and amino acids remobilization from green tissues to seeds.

Iron biofortification in rice: It's a long way to the top

Rice and most staple cereals contain low iron (Fe) levels, most of which is lost during grain processing. Populations with monotonous diets consisting mainly of cereals are especially prone to Fe deficiency, which affects about two billion people. Supplementation or food fortification programs have not always been successful. Crop Fe fertilization is also not very effective due to Fe soil insolubility. An alternative solution is Fe biofortification by generating cultivars that efficiently mobilize, uptake and translocate Fe to the edible parts. Here, we review the strategies used for the Fe biofortification of rice, including conventional breeding and directed genetic modification, which offer the most rapid way to develop Fe-rich rice plants. While classical breeding is able to modify the contents of inhibitors of Fe absorption, transgenic approaches have focused on enhanced Fe uptake from soil, xylem and phloem loading and grain sink strength. A comprehensive table is provided in which the percentages of the recommended dietary Fe intake reached by independently developed transgenic plants are calculated. In this review we also emphasize that the discovery of new QTLs and genes related to Fe biofortification is extremely important, but interdisciplinary research is needed for future success in this area.

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill

BackgroundEucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings.ResultsBy the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs indentified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm.ConclusionOur study showed that expression stability varied between putative reference genes tested in E. globulus. Based on the AGO1 relative expression profile obtained using the genes suggested by the algorithms, H2B and TUA were considered as the most suitable reference genes for expression studies in E. globulus adventitious rooting. UBI and 18S were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation.

Roles of plant metal tolerance proteins (MTP) in metal storage and potential use in biofortification strategies

Zinc (Zn) is an essential micronutrient for plants, playing catalytic or structural roles in enzymes, transcription factors, ribosomes, and membranes. In humans, Zn deficiency is the second most common mineral nutritional disorder, affecting around 30% of the worlds population. People living in poverty usually have diets based on milled cereals, which contain low Zn concentrations. Biofortification of crops is an attractive cost-effective solution for low mineral dietary intake. In order to increase the amounts of bioavailable Zn in crop edible portions, it is necessary to understand how plants take up, distribute, and store Zn within their tissues, as well as to characterize potential candidate genes for biotechnological manipulation. The metal tolerance proteins (MTP) were described as metal efflux transporters from the cytoplasm, transporting mainly Zn2+ but also Mn2+, Fe2+, Cd2+, Co2+, and Ni2+. Substrate specificity appears to be conserved in phylogenetically related proteins. MTPs characterized so far in plants have a role in general Zn homeostasis and tolerance to Zn excess; in tolerance to excess Mn and also in the response to iron (Fe) deficiency. More recently, the first MTPs in crop species have been functionally characterized. In Zn hyperaccumulator plants, the MTP1 protein is related to hypertolerance to elevated Zn concentrations. Here, we review the current knowledge on this protein family, as well as biochemical functions and physiological roles of MTP transporters in Zn hyperaccumulators and non-accumulators. The potential applications of MTP transporters in biofortification efforts are discussed.

Functional analysis of the rice vacuolar zinc transporter OsMTP1

Heavy metal homeostasis is maintained in plant cells by specialized transporters which compartmentalize or efflux metal ions, maintaining cytosolic concentrations within a narrow range. OsMTP1 is a member of the cation diffusion facilitator (CDF)/metal tolerance protein (MTP) family of metal cation transporters in Oryza sativa, which is closely related to Arabidopsis thaliana MTP1. Functional complementation of the Arabidopsis T-DNA insertion mutant mtp1-1 demonstrates that OsMTP1 transports Zn in planta and localizes at the tonoplast. When heterologously expressed in the yeast mutant zrc1 cot1, OsMTP1 complemented its Zn hypersensitivity and was also localized to the vacuole. OsMTP1 alleviated, to some extent, the Co sensitivity of this mutant, rescued the Fe hypersensitivity of the ccc1 mutant at low Fe concentrations, and restored growth of the Cd-hypersensitive mutant ycf1 at low Cd concentrations. These results suggest that OsMTP1 transports Zn but also Co, Fe, and Cd, possibly with lower affinity. Site-directed mutagenesis studies revealed two substitutions in OsMTP1 that alter the transport function of this protein. OsMTP1 harbouring a substitution of Leu82 to a phenylalanine can still transport low levels of Zn, with an enhanced affinity for Fe and Co, and a gain of function for Mn. A substitution of His90 with an aspartic acid completely abolishes Zn transport but improves Fe transport in OsMTP1. These amino acid residues are important in determining substrate specificity and may be a starting point for refining transporter activity in possible biotechnological applications, such as biofortification and phytoremediation.

Identification of Fe-excess-induced genes in rice shoots reveals a WRKY transcription factor responsive to Fe, drought and senescence.

Fe participates in several important reactions in plant metabolism. However, Fe homeostasis in plants is not completely understood, and molecular studies on Fe-excess stress are scarce. Rice (Oryza sativa L. ssp. indica) is largely cultivated in submerged conditions, where the extremely reductive environment can lead to severe Fe overload. In this work, we used representational difference analysis (RDA) to isolate sequences up-regulated in rice shoots after exposure to Fe-excess. We isolated 24 sequences which have putative functions in distinct cellular processes, such as transcription regulation (OsWRKY80), stress response (OsGAP1, DEAD-BOX RNA helicase), proteolysis (oryzain-α,rhomboid protein), photosynthesis (chlorophyll a/b binding protein), sugar metabolism (β glucosidase) and electron transport (NADH ubiquinone oxireductase). We show that the putative WRKY transcription factor OsWRKY80 is up-regulated in rice leaves, stems and roots after Fe-excess treatment. This up-regulation is also observed after dark-induced senescence and drought stress, indicating that OsWRKY80 could be a general stress-responsive gene. To our knowledge, this is the first report of an Fe-excess-induced transcription factor in plants.

Got to hide your Zn away: Molecular control of Zn accumulation and biotechnological applications

Zinc (Zn) is an essential micronutrient for all organisms, with key catalytic and structural functions. Zn deficiency in plants, common in alkaline soils, results in growth arrest and sterility. On the other hand, Zn can become toxic at elevated concentrations. Several studies revealed molecules involved with metal acquisition in roots, distribution within the plant and translocation to seeds. Transmembrane Zn transport proteins and Zn chelators are involved in avoiding its toxic effects. Plant species with the capacity to hyperaccumulate and hypertolerate Zn have been characterized. Plants that accumulate and tolerate high amounts of Zn and produce abundant biomass may be useful for phytoremediation, allowing cleaning of metal-contaminated soils. The study of Zn hyperaccumulators may provide indications of genes and processes useful for biofortification, for developing crops with high amounts of nutrients in edible tissues. Future research needs to focus on functional characterization of Zn transporters in planta, elucidation of Zn uptake and sensing mechanisms, and on understanding the cross-talk between Zn homeostasis and other physiological processes. For this, new research should use multidisciplinary approaches, combining traditional and emerging techniques, such as genome-encoded metal sensors and multi-element imaging, quantification and speciation using synchrotron-based methods.

Hypothyroidism changes adenine nucleotide hydrolysis in synaptosomes from hippocampus and cerebral cortex of rats in different phases of development

The influence of the thyroid hormones on the normal function of the mammalian central nervous system depends on the brain region and on the developmental stage. Adenine nucleotides and their products also affect the brain function; ATP is an excitatory neurotransmitter, and adenosine has inhibitory effects on neurotransmission. Thus, this study aimed to evaluate the effects of hypothyroidism on the hydrolysis of ATP to adenosine in hippocampal and cortical synaptosomes and blood serum of rats during different phases of development. Rats aged 60 and 420 days old were divided into three groups: control, sham‐operated and hypothyroid. Hypothyroidism was induced in these rats by thyroidectomy and methimazole (0.05%) added to their drinking water for 14 days. Neonatal hypothyroidism was induced by adding 0.02% methimazole in the drinking water from day 9 of gestation, and continually until 14 days old. Hypothyroidism increased the AMP hydrolysis in both hippocampus and cerebral cortex synaptosomes of rats in all aged tested. In blood serum, thyroid hormones deficiency increased the AMP hydrolysis in 14‐day‐old rats and the hydrolysis of ATP, ADP and AMP in 60‐day‐old rats; however, no alteration was observed in 420‐day‐old rats. Thus, our results suggest the involvement of the 5′‐nucleotidase in synaptic function control in hypothyroidism throughout brain development.

ZINC-INDUCED FACILITATOR-LIKE family in plants: lineage-specific expansion in monocotyledons and conserved genomic and expression features among rice ( Oryza sativa ) paralogs

BackgroundDuplications are very common in the evolution of plant genomes, explaining the high number of members in plant gene families. New genes born after duplication can undergo pseudogenization, neofunctionalization or subfunctionalization. Rice is a model for functional genomics research, an important crop for human nutrition and a target for biofortification. Increased zinc and iron content in the rice grain could be achieved by manipulation of metal transporters. Here, we describe the ZINC-INDUCED FACILITATOR-LIKE (ZIFL) gene family in plants, and characterize the genomic structure and expression of rice paralogs, which are highly affected by segmental duplication.ResultsSequences of sixty-eight ZIFL genes, from nine plant species, were comparatively analyzed. Although related to MSF_1 proteins, ZIFL protein sequences consistently grouped separately. Specific ZIFL sequence signatures were identified. Monocots harbor a larger number of ZIFL genes in their genomes than dicots, probably a result of a lineage-specific expansion. The rice ZIFL paralogs were named OsZIFL1 to OsZIFL13 and characterized. The genomic organization of the rice ZIFL genes seems to be highly influenced by segmental and tandem duplications and concerted evolution, as rice genome contains five highly similar ZIFL gene pairs. Most rice ZIFL promoters are enriched for the core sequence of the Fe-deficiency-related box IDE1. Gene expression analyses of different plant organs, growth stages and treatments, both from our qPCR data and from microarray databases, revealed that the duplicated ZIFL gene pairs are mostly co-expressed. Transcripts of OsZIFL4, OsZIFL5, OsZIFL7, and OsZIFL12 accumulate in response to Zn-excess and Fe-deficiency in roots, two stresses with partially overlapping responses.ConclusionsWe suggest that ZIFL genes have different evolutionary histories in monocot and dicot lineages. In rice, concerted evolution affected ZIFL duplicated genes, possibly maintaining similar expression patterns between pairs. The enrichment for IDE1 boxes in rice ZIFL gene promoters suggests a role in Zn-excess and Fe-deficiency up-regulation of ZIFL transcripts. Moreover, this is the first description of the ZIFL gene family in plants and the basis for functional studies on this family, which may play important roles in Zn and Fe homeostasis in plants.

Iron deficiency in rice shoots: identification of novel induced genes using RDA and possible relation to leaf senescence.

Rice plants are highly susceptible to Fe-deficiency. Under nutrient deprivation, plant cells undergo extensive metabolic changes for their continued survival. To provide further insight into the pathways induced during Fe-deficiency, rice seedlings were grown for 3, 6 and 9 days in the presence or absence of Fe. Using RDA (Representational Difference Analysis), sequences of 32 induced genes in rice shoots under Fe-deficiency were identified. About 30% of the sequences found have been previously reported as responsive to other abiotic and even biotic stresses. However, this is the first report that indicates their relation to Fe deprivation. Differential expression of selected genes was confirmed by semi-quantitative RT-PCR analysis. The identification of classical senescence-related sequences, such as lipase EC 3.1.1.-, ubiquitin-conjugating enzyme EC 6.3.2.19, β-Glucosidase EC 3.2.1.21 and cysteine synthase EC 2.5.1.47, besides the higher accumulation of total soluble sugars prior to the decrease of total chlorophyll content in Fe-deficient leaves, indicate that sugar accumulation may be one of the factors leading to premature leaf senescence induced by Fe-deficiency.