Raul Antonio Sperotto

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.

Identification of putative target genes to manipulate Fe and Zn concentrations in rice grains.

Rice is the staple food of half of the worlds population; however, it is a poor source of essential micronutrients such as Fe and Zn. Since flag leaves are one of the sources of remobilized metals for developing seeds, the identification of the molecular players that might contribute to the process of metal transport from flag leaves to the seeds may be useful for biofortification purposes. We analyzed the expression of 25 metal-related genes from rice, including rice homologues for YSLs, NRAMPs, ZIPs, IRT1, VIT1 (coding for known or potential metal transporters), as well as NASs, FROs and NAC5 (involved in metal homeostasis) in flag leaves of eight rice cultivars (showing contrasting levels of seed Fe and Zn) during panicle emergence (R3) and grain filling stage (R5). The expression level of nine of these genes (OsYSL6, OsYSL8, OsYSL14, OsNRAMP1, OsNRAMP7, OsNRAMP8, OsNAS1, OsFRO1 and OsNAC5) in flag leaves exhibited significant correlations with Fe and/or Zn concentrations in the seeds. In this way, our study has provided a short list of putative target genes to manipulate Fe and Zn concentrations in rice grains.

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.

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.

Effects of different Fe supplies on mineral partitioning and remobilization during the reproductive development of rice (Oryza sativa L.)

BackgroundMinimal information exists on whole-plant dynamics of mineral flow through rice plants and on the source tissues responsible for mineral export to developing seeds. Understanding these phenomena in a model plant could help in the development of nutritionally enhanced crop cultivars. A whole-plant accumulation study, using harvests during reproductive development under different Fe supplies, was conducted to characterize mineral accumulation in roots, non-flag leaves, flag leaves, stems/sheaths, and panicles of Kitaake rice plants.ResultsLow Fe supply promoted higher accumulation of Zn, Cu and Ni in roots, Mn, Ca, Mg and K in leaves and Zn in stems/sheaths and a smaller accumulation of Fe, Mn and Ca in roots and Zn and Ni in leaves. High Fe supply promoted higher accumulation of Fe in roots and Zn in leaves and a smaller accumulation of Fe in leaves and stems/sheaths and Zn, Cu and K in roots. Correlation analyzes indicated that fluctuations in Mn-Ca, Zn-Cu, Zn-Ni, Cu-Ni, Mo-S, Ca-Mg, Cu-Mn and Cu-Mg concentrations in response to different Fe supplies were positively correlated in at least four of the five organs analyzed.ConclusionsMineral content loss analysis indicated that mineral remobilization from vegetative organs can occur in rice plants; however, for seeds to acquire minerals, vegetative remobilization is not absolutely required. Also, mineral remobilization from vegetative tissues in rice was greatly dependent of plant Fe nutrition. Remobilization was observed for several minerals from flag leaves and stems/sheaths, but the amounts were generally far below the total mineral accretion observed in panicles, suggesting that continued uptake and translocation of minerals from the roots during seed fill are probably more important than mineral remobilization.

Influence of iron on mineral status of two rice (Oryza sativa L.) cultivars

Received: 02 April 2007; Returned for revision: 02 July 2007; Accepted: 14 August 2007Iron is an essential nutrient for plants. In aerobic conditions, Fe is highly unavailable for plant uptake, and Fe deficiencycan be severe in plants grown in calcareous soils. In waterlogged soils, however, Fe availability increases and can reachtoxic concentrations. Rice is an important staple crop worldwide and faces iron deficiency or excess, depending on thegrowth conditions. To contribute to the study of mechanisms involved in response to Fe deficiency and resistance to Feexcess, experiments were carried out with rice cultivars BR-IRGA 409 (I409, susceptible to Fe toxicity) and EPAGRI 108(E108, resistant to Fe toxicity) grown in culture solutions and submitted to Fe excess, control concentration ordeficiency (500, 6.5 or zero mg L

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.

Zn/Fe remobilization from vegetative tissues to rice seeds: should I stay or should I go? Ask Zn/Fe supply!

Rice is a staple food for at least 50% of the worlds population. Therefore, it is one of the most important crop plants on Earth (Lucca et al., 2002). However, milled rice is a poor source of essential micronutrients such as Fe and Zn (Bouis and Welch, 2010), whose deficiencies affect over three billion people worldwide, mostly in developing countries (Welch and Graham, 2004). Fe and Zn malnutrition are leading risk factors for disability and death, especially to children eating cereal-based diets, resulting in impaired functions of the brain, the immune and reproductive systems and energy metabolism (Graham et al., 2001). The development of new cultivars with elevated concentrations of Fe and Zn would be extremely relevant to alleviate malnutrition, but the lack of knowledge about how nutrients are translocated from vegetative tissues to the seeds is one of the barriers to rice biofortification (Colangelo and Guerinot, 2006; Sperotto et al., 2012a).