Marta W. Vasconcelos

Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene

In this report, we show that the expression of the soybean ferritin gene, driven by the endosperm-specific glutelin promoter, leads to higher iron and zinc levels in transgenic indica rice grains. Brown rice is rarely consumed, and polishing of the rice grain brings considerable loss of micronutrients by removing its outer layers. No data until now have shown that after commercial milling the micronutrient concentration remains higher than that of the control. In our experiment, expression of the soybean ferritin gene under the control of the glutelin promoter in rice has proven to be effective in enhancing grain nutritional levels, not only in brown grains but also in polished grains. Besides determining the iron levels in transgenic rice grains, we also checked for zinc concentration, and it was found to be higher in transgenic seeds than in the control. Moreover, we introduced this gene in an elite indica rice line that has highly desirable agronomic and field-performance traits. Prussian blue staining reaction clearly revealed the presence of iron in the endosperm cells of transgenic rice grains, and immunolocalization revealed the presence of the expression gene in the endosperm of the transgenic material. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Chemical composition of red, brown and green macroalgae from Buarcos bay in Central West Coast of Portugal.

Six representative edible seaweeds from the Central West Portuguese Coast, including the less studied Osmundea pinnatifida, were harvested from Buarcos bay, Portugal and their chemical characterization determined. Protein content, total sugar and fat contents ranged between 14.4% and 23.8%, 32.4% and 49.3% and 0.6-3.6%. Highest total phenolic content was observed in Codium tomentosum followed by Sargassum muticum and O. pinnatifida. Fatty acid (FA) composition covered the branched chain C13ai to C22:5 n3 with variable content in n6 and n3 FA; low n6:n3 ratios were observed in O. pinnatifida, Grateloupia turuturu and C. tomentosum. Some seaweed species may be seen as good sources of Ca, K, Mg and Fe, corroborating their good nutritional value. According to FTIR-ATR spectra, G. turuturu was associated with carrageenan seaweed producers whereas Gracilaria gracilis and O. pinnatifida were mostly agar producers. In the brown algae, S. muticum and Saccorhiza polyschides, alginates and fucoidans were the main polysaccharides found.

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.

Characterization of the PT Clade of Oligopeptide Transporters in Rice

Oligopeptide transporters (OPTs) are a group of membrane‐localized proteins that have a broad range of substrate transport capabilities and that are thought to contribute to many biological processes. Nine OPTs belonging to the peptide transport (PT) clade were identified in the rice (Oryza sativa L.) genome, based on their predicted amino acid sequences containing the two highly conserved motifs (NPG and KIPPR) that define the PT‐OPT subgroup. Protein identity analysis comparing the rice OPTs of the PT and yellow stripe (YS) clades revealed low levels of similarity between members of these two OPT subgroups. Sequence analysis of the predicted proteins of the PT clade, named OsOPT1 to OsOPT9, revealed all to be integral membrane proteins. Functional analysis of five of the OsOPTs, using a yeast complementation assay, demonstrated that OsOPT1, OsOPT3, OsOPT4, OsOPT5, and OsOPT7 could transport ferrous and/or ferric iron chelated to nicotianamine, a nonpeptidyl metal‐chelate. Semi quantitative reverse‐transcriptase polymerase chain reaction verified expression of the OsOPTs in roots, stems, leaves, hulls, pedicels, and embryos, but transcripts for each OsOPT were not found in every tissue. The identification of multiple PT‐clade OPT genes in rice with different expression patterns indicates that they may have different biological roles in the plant.

Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening.

BackgroundPine wilt disease (PWD), caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus), damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant’s molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN) and Pinus pinea (less susceptible to PWN).ResultsFour cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species.ConclusionsDefense-related genes triggered by nematode infestation were detected in both P. pinaster and P. pinea transcriptomes utilizing 454 pyrosequencing technology. P. pinaster showed higher abundance of genes related to transcriptional regulation, terpenoid secondary metabolism (including some with nematicidal activity) and pathogen attack. P. pinea showed higher abundance of genes related to oxidative stress and higher levels of expression in general of stress responsive genes. This study provides essential information about the molecular defense mechanisms utilized by P. pinaster and P. pinea against PWN infestation and contributes to a better understanding of PWD.

Population dynamics of bacteria associated with different strains of the pine wood nematode Bursaphelenchus xylophilus after inoculation in maritime pine (Pinus pinaster)

For a long time it was thought that Bursaphelenchus xylophilus was the only agent of the pine wilt disease. Recently, it was discovered that there are bacteria associated with the nematodes that contribute to the pathogenesis of this disease, mainly through the release of toxins that promote the death of the pines. Among the species most commonly found, are bacteria belonging to the Bacillus, Pantoea, Pseudomonas and Xanthomonas genera. The main objective of this work was to study the effect of inoculation of maritime pine (Pinus pinaster) with four different nematode isolates, in the bacterial population of nematodes and trees, at different stages of disease progression. The monitoring of progression of disease symptoms was also recorded. Also, the identification of bacteria isolated from the xylem of trees and the surface of nematodes was performed by classical identification methods, by the API20E identification system and by sequencing of bacterial DNA. The results showed that for the symptoms progression, the most striking difference was observed for the pines inoculated with the avirulent isolate, C14-5, which led to a slower and less severe aggravation of symptoms than in pines inoculated with the virulent isolates. In general, it was found that bacterial population, inside the tree, increased with disease progression. A superior bacterial quantity was isolated from pines inoculated with the nematode isolates HF and 20, and, comparatively, few bacteria were isolated from pines inoculated with the avirulent isolate. The identification system API20E was insufficient in the identification of bacterial species; Enterobacter cloacae species was identified in 79% of the isolated bacterial colonies and seven of these colonies could not be identified by this method. Molecular identification methods, through bacterial DNA sequencing, allowed a more reliable identification: eleven different bacterial species within the Bacillus, Citrobacter, Enterobacter, Escherichia, Klebsiella, Paenibacillus, Pantoea and Terribacillus genera were identified. General bacterial diversity increased with the progression of the disease. Bacillus spp. were predominant at the earlier stage of disease progression and Klebsiella oxytoca at the later stages. Furthermore, bacterial species isolated from the surface of nematodes were similar to those isolated from the xylem of pines. In the present work new bacterial species were identified which have never been reported before in this type of study and may be associated with their geographical origin (Portugal). P. pinaster, the pine species used in this study, was different from those commonly grown in Japan and China. Furthermore, it was the first time that bacteria were isolated and identified from an avirulent pine wood nematode isolate.

Functional analysis of transgenic Rice (Oryza sativa L.) transformed with an Arabidopsis thaliana ferric reductase (AtFRO2).

Abstract Iron deficient soils limit crop production on 25-30% of the worlds arable land. Both grasses (Strategy 11) and dicotyledonous crops (Strategy 1) are susceptible to iron deficiency, but each respond to iron stress by different mechanisms. In order to acquire iron from the soil, Strategy I plants utilize an iron reduction and Fe2+ transporter system at the root level, whereas Strategy 11 plants use a phytosiderophore-based system. Unfortunately, in some grasses such as rice, the production of phytosiderophores is low, and thus their ability to survive in iron-deficient conditions is limited. To determine whether a Strategy I root reductase can function in a Strategy 11 plant, and enhance its iron acquisition, we inserted the FRO2 gene from Arabidopsis thaliena (AtFR02) into rice (Oryza sativa). Root reductase activity was determined and was found to be low in both transgenic and control plants grown at different iron concentrations. The low activity levels were attributed to the release of soluble reductants in the assay and not to membrane-localized root reductase activity. RT-PCR analysis of rice roots and shoots of plants grown hydroponically at different iron concentrations revealed no expression of the transgene. In this paper, we discuss the lack of functionality of the AtFRO2 gene in rice, and we perform a comparative study of the 0.6 kb promoter region by PlantCARE and PLACE analysis.

Morpho-physiological parameters affecting iron deficiency chlorosis in soybean ( Glycine max L.)

Background and aimsIron deficiency chlorosis (IDC) leads to severe leaf chlorosis, low photosynthetic rates, and yield reductions of several million metric tonnes each year. In order to devise breeding and genetic transformation programs that aim at generating high-yielding and IDC-tolerant soybean lines, it is necessary to better understand the mechanisms that enable tolerant plants to survive under Fe-limiting conditions.MethodsAn in silico analysis in the USDA soybean collection allowed the identification of a set of novel efficient and inefficient soybean cultivars which can be used in future studies concerning IDC response. Plants were grown in iron deficient and iron sufficient conditions using a bicarbonate system and several IDC-related aspects were studied.ResultsA new set of efficient and inefficient soybean lines were identified in silico, and their tolerance to IDC was confirmed under laboratorial conditions. New plant traits that are highly correlated to IDC scoring were identified: a negative correlation was found between SPAD values and stem weight, weight of the unifoliolates and iron concentration of the first unifoliolates was found; higher SPAD values were correlated with the amount of iron in the first trifoliate leaves. Our data also show that having higher concentrations of iron in the seeds provides increased resistance to IDC. No correlation was found between root iron reductase activity and chlorosis.ConclusionsSoybean differential chlorosis susceptibility between different accessions is linked to specific morpho-physiological parameters such as unifoliolate leaf size, stem weigh, concentration of iron in the seeds, and tissue iron partitioning.

Evaluation of chitooligosaccharide application on mineral accumulation and plant growth in Phaseolus vulgaris

Chitooligosaccharides (COS) - water soluble derivatives from chitin, are an interesting group of molecules for several biological applications, for they can enter plant cells and bind negatively charged molecules. Several studies reported an enhanced plant growth and higher crop yield due to chitosan application in soil grown plants, but no studies have looked on the effect of COS application on plant mineral nutrient dynamics in hydroponically grown plants. In this study, Phaseolus vulgaris was grown in hydroponic culture and the effect of three different concentrations of COS on plant growth and mineral accumulation was assessed. There were significant changes in mineral allocations for Mo, B, Zn, P, Pb, Cd, Mn, Fe, Mg, Ca, Cu, Na, Al and K among treatments. Plant morphology was severely affected in high doses of COS, as well as lignin concentration in the stem and the leaves, but not in the roots. Chlorophyll A, B and carotenoid concentrations did not change significantly among treatments, suggesting that even at higher concentrations, COS application did not affect photosynthetic pigment accumulation. Plants grown at high COS levels had shorter shoots and roots, suggesting that COS can be phytotoxic to the plant. The present study is the first detailed report on the effect of COS application on mineral nutrition in plants, and opens the door for future studies that aim at utilizing COS in biofortification or phytoremediation programs.

Iron biofortification in the 21st century: setting realistic targets, overcoming obstacles, and new strategies for healthy nutrition

Plant-based foods offer a wide range of nutrients that are essential for human and animal health. Among these nutrients, iron stands out as one of the most important micronutrients. Increasing the iron content in many staple and non-staple plant foods continues to be a goal of many scientists around the world. However, the success of such initiatives has sometimes fallen short of their expected targets. In this review we highlight the most recent and promising results that have contributed to increasing the iron content in different crops. We also discuss methods that to date have been used to reach iron biofortification goals and new strategies that we believe are most promising for crop biofortification in the future. Plant anatomical, physiological and metabolic hurdles still need to be tackled for making progress on further increasing currently reached levels of micronutrient improvements. New strategies need to take into account growing environmental challenges that may constrain biofortification efforts.