Genetic Engineering Of Crop Plants Research Articles

Tissue-specific expression of Ruby in Mexican lime (C. aurantifolia) confers anthocyanin accumulation in fruit.

Tissue specific promoters are important tools for the precise genetic engineering of crop plants. Four fruit-preferential promoters were examined for their ability to confer a novel fruit trait in transgenic Mexican lime (Citrus aurantifolia). The Ruby transcription factor activates fruit anthocyanin accumulation within Moro blood orange and has been shown to function in activating anthocyanin accumulation in heterologous plant species. Although the CitVO1, CitUNK, SlE8, and PamMybA promoters were previously shown to confer strong fruit-preferential expression in transgenic tomato, they exhibited no detectable expression in transgenic Mexican lime trees. In contrast, the CitWax promoter exhibited high fruit-preferential expression of Ruby, conferring strong anthocyanin accumulation within the fruit juice sac tissue and moderate activity in floral/reproductive tissues. In some of the transgenic trees with high levels of flower and fruit anthocyanin accumulation, juvenile leaves also exhibited purple coloration, but the color disappeared as the leaves matured. We show that the CitWax promoter enables the expression of Ruby to produce anthocyanin colored fruit desired by consumers. The production of this antioxidant metabolite increases the fruits nutritional value and may provide added health benefits.

Coordination Between Phloem Loading and Structure Maintains Carbon Transport Under Drought.

Maintaining phloem transport under water stress is expected to be crucial to whole-plant drought tolerance, but the traits that benefit phloem function under drought are poorly understood. Nearly half of surveyed angiosperm species, including important crops, use sucrose transporter proteins to actively load sugar into the phloem. Plants can alter transporter abundance in response to stress, providing a potential mechanism for active-loading species to closely regulate phloem loading rates to avoid drought-induced reductions or failures in phloem transport. We developed an integrated xylem-phloem-stomatal model to test this hypothesis by quantifying the joint impacts of transporter kinetics, phloem anatomy, and plant water status on sucrose export to sinks. We parameterized the model with phloem hydraulic resistances and sucrose transporter kinetic parameters compiled from the literature, and simulated loading regulation by allowing loading rates to decline exponentially with phloem pressure to prevent excessive sucrose concentrations from inducing viscosity limitations. In the absence of loading regulation, where loading rates were independent of phloem pressure, most resistance values produced unrealistic phloem pressures owing to viscosity effects, even under well-watered conditions. Conversely, pressure-regulated loading helped to control viscosity buildup and improved export to sinks for both lower and higher resistant phloem pathways, while maintaining realistic phloem pressures. Regulation also allowed for rapid loading and export in wet conditions while maintaining export and viable phloem pressures during drought. Therefore, we expect feedbacks between phloem pressure and loading to be critical to carbon transport in active-loading species, especially under drought, and for transporter kinetics to be strongly coordinated with phloem architecture and plant water status. This work provides an important and underexplored physiological framework to understand the ecophysiology of phloem transport under drought and to enhance the genetic engineering of crop plants.

Harmony but Not Uniformity: Role of Strigolactone in Plants.

Strigolactones (SLs) represent an important new plant hormone class marked by their multifunctional roles in plants and rhizosphere interactions, which stimulate hyphal branching in arbuscular mycorrhizal fungi (AMF) and seed germination of root parasitic plants. SLs have been broadly implicated in regulating root growth, shoot architecture, leaf senescence, nodulation, and legume–symbionts interaction, as well as a response to various external stimuli, such as abiotic and biotic stresses. These functional properties of SLs enable the genetic engineering of crop plants to improve crop yield and productivity. In this review, the conservation and divergence of SL pathways and its biological processes in multiple plant species have been extensively discussed with a particular emphasis on its interactions with other different phytohormones. These interactions may shed further light on the regulatory networks underlying plant growth, development, and stress responses, ultimately providing certain strategies for promoting crop yield and productivity with the challenges of global climate and environmental changes.

Transgenic approaches for plant disease control: Status and prospects 2021

AbstractPlant diseases represent a major constraint on agricultural production. Finding sustainable novel means for their control is an important challenge. The ever‐increasing knowledge and understanding of plant–microbe interactions has led to several ingenious transgenic approaches to combat disease. The first transgenic disease‐resistant plants expressed single or a few stacked genes encoding antimicrobial proteins. Whereas the first attempts were disappointing in the field, several examples from recent field studies are promising and some of these use ingenious designer approaches. Less progress has been made with antimicrobial metabolites where the challenges lie in obtaining biosynthetic genes and in coordinating their expression. The increased understanding of the processes regulating plant defence (plant immunity) and modes of action of pathogen effector proteins have also led to novel strategies for designing resistant plants. The most promising of these is host‐induced gene silencing that targets specific pathogens, either the effectors or, preferably, essential housekeeping genes. With these approaches, and several maverick examples of “genes pulled out of a hat,” the technical effort in designing resistant plants is finally paying off. The prospects are good, biologically speaking, but can industry deliver? There is still an issue of public acceptance of genetic engineering of crop plants, especially in Europe; so whilst considerable strategic and practical progress has been made over the last decade, vanishingly few products have been adopted by agriculture. Some of these have been in use for over two decades. As yet, all are against viral diseases and not against diseases caused by microorganisms.

An Apical Meristem-Targeted in planta Transformation Method for the Development of Transgenics in Flax (Linum usitatissimum): Optimization and Validation.

Efficient regeneration of explants devoid of intrinsic somaclonal variations is a cardinal step in plant tissue culture, thus, a vital component of transgenic technology. However, recalcitrance of economically important crops to tissue culture-based organogenesis ensues a setback in the use of transgenesis in the genetic engineering of crop plants. The present study developed an optimized, genotype-independent, nonconventional tissue culture-independent in planta strategy for the genetic transformation of flax/linseed. This apical meristem-targeted in planta transformation protocol will accelerate value addition in the dual purpose industrially important but recalcitrant fiber crop flax/linseed. The study delineated optimization of Agrobacterium tumefaciens-mediated transformation and stable T-DNA (pCambia2301:GUS:nptII) integration in flax. It established successful use of a stringent soilrite-based screening in the presence of 30 mg/L kanamycin for the identification of putative transformants. The amenability, authenticity, and reproducibility of soilrite-based kanamycin screening were further verified at the molecular level by GUS histochemical analysis of T0 seedlings, GUS and nptII gene-specific PCR, genomic Southern hybridization for stable integration of T-DNA, and expression analysis of transgenes by sqRT-PCR. This method resulted in a screening efficiency of 6.05% in the presence of kanamycin, indicating amenability of in planta flax transformation. The strategy can be a promising tool for the successful development of transgenics in flax.

Integrating expression data and genomic sequences to investigate transcriptional regulation in barley in response to abiotic stress.

Abiotic stress responses are regulated critically at the transcriptional level. Clarifying the intricate mechanisms that regulate gene expression in response to abiotic stress is crucial and challenging. For this purpose, the factors that regulate gene expression and their binding sites in DNA should be determined. By using bioinformatics tools, the differentially expressed probe sets were studied. A meta-analysis of transcriptomic responses to several abiotic stresses in barley was performed. Motif enrichments revealed that AP2/ERF (APETALA2/Ethylene-Responsive Factor) has the most frequent binding sites. We found that the bHLH transcription factor family has the highest number of transcription factor members. Moreover, network construction revealed that AP2 has the highest number of connections with other genes, which indicates its critical role in abiotic stress responses. The present research further predicted 49 miRNAs belonging to 23 miRNA families. This study identified the probable conserved and enriched motifs, which might have a role in the regulation of differentially expressed genes under abiotic stresses. In addition to shedding light on gene expression regulation, a toolbox of available promoters for genetic engineering of crop plants under such abiotic stresses was developed.

Transcriptomic analysis reveals somatic embryogenesis-associated signaling pathways and gene expression regulation in maize (Zea mays L.).

Transcriptome analysis of maize embryogenic callus and somatic embryos reveals associated genes reprogramming, hormone signaling pathways and transcriptional regulation involved in somatic embryogenesis in maize. Somatic embryos are widely utilized in propagation and genetic engineering of crop plants. In our laboratory, an elite maize inbred line Y423 that could generate intact somatic embryos was obtained and applied to genetic transformation. To enhance our understanding of regulatory mechanisms during maize somatic embryogenesis, we used RNA-based sequencing (RNA-seq) to characterize the transcriptome of immature embryo (IE), embryogenic callus (EC) and somatic embryo (SE) from maize inbred line Y423. The number of differentially expressed genes (DEGs) in three pairwise comparisons (IE-vs-EC, IE-vs-SE and EC-vs-SE) was 5767, 7084 and 1065, respectively. The expression patterns of DEGs were separated into eight major clusters. Somatic embryogenesis associated genes were mainly grouped into cluster A or B with an expression trend toward up-regulation during dedifferentiation. GO annotation and KEGG pathway analysis revealed that DEGs were implicated in plant hormone signal transduction, stress response and metabolic process. Among the differentially expressed transcription factors, the most frequently represented families were associated with the common stress response or related to cell differentiation, embryogenic patterning and embryonic maturation processes. Genes include hormone response/transduction and stress response, as well as several transcription factors were discussed in this study, which may be potential candidates for further analyses regarding their roles in somatic embryogenesis. Furthermore, the temporal expression patterns of candidate genes were analyzed to reveal their roles in somatic embryogenesis. This transcriptomic data provide insights into future functional studies, which will facilitate further dissections of the molecular mechanisms that control maize somatic embryogenesis.

Functional characterization of wheat myo-inositol oxygenase promoter under different abiotic stress conditions in Arabidopsis thaliana.

The production of wheat is severely affected by abiotic stresses such as cold, drought, salinity, and high temperature. Although constitutive promoters are frequently used to regulate the expression of alien genes, these may lead to undesirable side-effects in transgenic plants. Therefore, identification and characterization of an inducible promoter that can express transgene only when exposed to stresses are of great importance in the genetic engineering of crop plants. Previous studies have indicated the abiotic stress-responsive behavior of myo-inositol oxygenase (MIOX) gene in different plants. Here, we isolated the MIOX gene promoter from wheat (TaMIOX). The in-silico analysis revealed the presence of various abiotic stress-responsive cis-elements in the promoter region. The TaMIOX promoter was fused with the UidA reporter gene and transformed into Arabidopsis thaliana. The T3 single-copy homozygous lines were analyzed for GUS activity using histochemical and fluorometric assays. Transcript expression of TaMIOX::UidA was significantly up-regulated by heat (fivefold), cold (sevenfold), and drought (fivefold) stresses as compared to transgenic plants grown without stress-induced conditions. The CaMV35S::UidA plants showed very high GUS activity even in normal conditions. In contrast, the TaMIOX::UidA plants showed prominent GUS activity only in stress treatments (cold, heat, and drought), which suggests the inducible behavior of the TaMIOX promoter. The substrate myo-inositol feeding assay of TaMIOX::UidA plants showed lesser GUS activity as compared to plants treated in abiotic stress conditions. Results support that the TaMIOX promoter could be used as a potential candidate for conditional expression of the transgene in abiotic stress conditions.

Cold-inducible expression of an Arabidopsis thaliana AP2 transcription factor gene, AtCRAP2, promotes flowering under unsuitable low-temperatures in chrysanthemum

Flowering time is regulated by biotic and abiotic stresses and affected by the ambient temperature. For chrysanthemum, a low ambient growth temperature can cause a flowering delay, which limits the annual commercial production. Therefore, it is important to improve the low-temperature flowering capability of chrysanthemum through genetic modifications. Here, we isolated a natural variation of a CRT/DRE-binding factor (CBF/DREB) 3 gene, CRAP2, from the Arabidopsis thaliana accession Condara (190AV) that encodes a stop codon at position 151 of the CBF3 protein. Unlike AtCBF3, the overexpression AtCRAP2 in Arabidopsis did not cause detectable growth retardation nor delayed flowering and it conferred cold tolerance. The cold-inducible expression of AtCRAP2 in chrysanthemum promoted flowering under short-day conditions with a low 15 °C nighttime temperature. RNA-sequencing of rd29A:AtCRAP2 and qRT-PCR assays of flowering time-related genes and AtCRAP2 expressed at an ambient temperature revealed that AtCRAP2 positively affected SOC1 and FTL3, thereby promoting flowering under low temperature stress and short-day conditions. These results indicate that DREB genes can be used in the genetic engineering of crop plants without accompanying negative effects by modifying the encoded proteins’ C termini.

An apple transcription factor, MdDREB76, confers salt and drought tolerance in transgenic tobacco by activating the expression of stress-responsive genes.

An apple gene, MdDREB76 encodes a functional transcription factor and imparts salinity and drought stress endurance to transgenic tobacco by activating expression of stress-responsive genes. The dehydration-responsive element (DRE)-binding protein (DREB) transcription factors are well known to be involved in regulating abiotic stress-mediated gene expression in plants. In this study, MdDREB76 gene was isolated from apple (Malus x domestica), which encodes a functional transcription factor protein. Overexpression of MdDREB76 in tobacco conferred salt and drought stress tolerance to transgenic lines by inducing antioxidant enzymes, such as superoxide dismutase, ascorbate peroxidase and catalase. The higher membrane stability index, relative water content, proline, total soluble sugar content and lesser H2O2content, electrolyte leakage and lipid peroxidation in transgenics support the improved physiological status of transgenic plants as compared to WT plants under salinity and drought stresses. The MdDREB76 overexpression upregulated the expression of stress-responsive genes that provide salinity and drought stress endurance to the plants. Compared to WT plants, transgenic lines exhibited healthy growth and higher yield under stress conditions. The present study reports MdDREB76 as a key regulator that switches on the battery of downstream genes which impart salt and osmotic stress endurance to the transgenic plants and can be used for genetic engineering of crop plants to combat salinity and drought stresses.

Ethical Dilemma with Respect to CBD Regulations in Genetic Modification of Biological Resources in Cameroon

The quest for patent rights has seen bioprospecting as a scientific and commercial research paradigm in which bioprospectors explore secluded locations around Cameroon in order to find ‘new drugs from exotic plants’. Bioprospectors derive genetic and biochemical materials that are both scientifically and commercially valuable, and they subsequently patent these materials abroad away from the original source to justify legal ownership through intellectual property law. An almost unprecedented amount of discussion has been stimulated on the merits and demerits of genetic engineering of crop plants and biodiversity exploitation and has divided both the public and scientific communities. The arguments for and against genetic engineering are invariably based on visions or missions of the new technology from widely different ethical perspectives. Fundamental issues of man's relationship with nature and theological matters are issues of concern. The genetic engineering of living cells, plants, animals and human beings has brought ethical concerns and issues to the conservation of biodiversity. Agricultural productivity depends in part on the availability of biodiversity for the development of improved cultivars. Until the 1970s, biodiversity was considered to be part of the ‘common heritage of humanity’. Under the regime of patent rights, biological resources are treated as belonging to the ‘public domain’ and are not owned by any individual, group, or state. From a common heritage of mankind, biodiversity is evolving into a resource under the sovereignty of nation states and is subject to intellectual property rights (IPRs). The new technology has witnessed a lot of illegal exploitation and commercialisation of these biological resources which is considered as biopiracy.

Isolation and functional characterization of a novel stress inducible promoter from pigeonpea (Cajanus cajan L)

The present study deals with isolation and characterization of a novel hybrid-proline-rich protein gene (CcHyPRP) promoter from pigeonpea. Real time PCR analysis revealed that CcHyPRP expression was strongly induced by dehydration, salt, Abscisic acid (ABA) and Salicylic acid (SA) treatments. The CcHyPRP promoter, isolated by genome-walking method, contained 1112 bp and showed the presence of various cis-regulatory elements necessary for tissue specific expression and stress responsiveness. Different 5′ deletions of the promoter were generated and were used to drive the expression of β-glucuronidase reporter gene (gusA) in Arabidopsis thaliana. Histochemical and fluorometric assays confirmed that GUS expression driven by the full-length fragment (1112 bp) was higher when compared to different deletion fragments. Under normal conditions, GUS expression was predominantly detected in the roots and hypocotyls of transformants, while under mannitol, NaCl, ABA and SA treatment conditions higher GUS expression levels were observed in the roots and leaves. However, the GUS expression was mostly confined to the roots of transformants carrying 477 and 300 bp promoter regions. The results amply indicate that CcHyPRP promoter is regulated by different stress factors, and as such the promoter can be deployed in genetic engineering of crop plants for enhanced abiotic stress tolerance.

Genetic Engineering of Crop Plants for Drought Tolerance:Role of Transcription Factors

Agricultural production is severely affected by various abiotic stresses including drought. Development of crop plants tolerant to drought is an urgent need to increase crop productivity to feed the growing population. It is imperative to understand the adaptive mechanism of plants to drought especially the type and expression levels of drought responsive genes. Several genes upregulated during abiotic stress have been exploited to develop drought tolerant plants. Genetic engineering of crop plants with stress responsive genes has been an effective method of generating drought tolerant plants. But expressions of these stress responsive genes are regulated by several transcription factors (TFs). TFs are DNA binding proteins that bind to the specific sequence motif and modulate the levels of gene expression. They may function as an activator, or repressor for the expression of specific genes. Transgenic plants expressing drought responsive TFs have been shown to improve drought tolerance in many crop plants. In this review, we have discussed the details on TFs used for developing various transgenic plants and also analyzed the potential of TFs for further transgenic studies. The future direction to exploit TFs for sustainable crop production under drought is also highlighted.

Special Issue on Sustainable Agriculture under Abiotic and Biotic Stress

World poverty and hunger are closely related toenvironmental degradation, caused by increasingpopulation pressure and urban expansions, soilerosion, limitation of water resources, gaseouspollution, and massive industrialization.Furthermore, climate changes and extreme weatherpatterns pose new challenges for crop productionand food security. Sustainable agriculture for theproduction of food and fiber, usingenvironmentally-conscious farming practices basedon understanding plant ecological interactions, iscrucial for the preservation of natural resources forfuture generations.This special issue focuses on recent researchprogress on understanding plant interaction withabiotic and biotic stress factors for sustainableagriculture. Plant stresses inflected by salinity,drought, floods, temperature extremes, radiation,and toxic substances deposited in the soil togetherwith diseases and insects attacks, are the majorlimitations of crop cultivation incurring substantialreduction in yield and hence elevate economiclosses to the farmers.Sustainable agriculture is challenged by thelosses in crop genetic diversity. Conservation ofavailable germplasm and the efficient utilization ofgenetic resources are important aspects of currentresearch. In this special issue, Yumurtaci (2015)reviewed the utilization of wild relatives for thedevelopment of abiotic and biotic stress tolerantnew varieties of four major food crops includingwheat, barley, maize and oat.Modern advances in biotechnology offerinnovative approaches for understanding thefundamental mechanisms of abiotic and bioticstress and the development of stress tolerant crops.On this topic, Bakhsh (2015) reviewed currentresearch achievements towards the development ofabiotic stress tolerant crops through geneticengineering. In addition, Ram and Sharma (2015)studied the molecular characterization of TaPase inwheat, a gene known to be involved in plantresponse to stress.Plant diseases represent another major challengefor sustainable agriculture. To illustrate theimportance of integrating genetic engineering in thestrategies to combat plant diseases, Elayabalan etal. (2015) reviewed relevant advances in thedevelopment of banana plants resistant to thebanana bunchy top disease.Moreover, crop plants must adapt to adverseclimatic conditions, particularly those associatedwith the global climatic changes. Stresses due toabiotic stress factors like drought, extremetemperature and salinity and heavy metal toxicityinflect tremendous losses in crop productivity andare considered as major challenging factors forfuture agriculture sustainability. This topic wasaddressed in this special issue in several papers.Ud-Din et al. (2015) addressed drought effect onrice yield in relation to hormonal application.Siddika et al. (2015) studied the response to hightemperature stress of Basella alba, a leaf vegetablecommonly names as vine spinach.On the topic of heavy metal toxicity, Fasahat(2015) reviewed progress made in understandingcadmium toxicity and tolerance in rice. The specialissue also include studies on nutrient uptake fromunder saline conditions in potato (Oustani et al.,2015) and ion content in wild sage (Lantanacamara) treated with mepiquat chloride applicationunder shading (Matsoukis et al., 2015). In additionAjambang et al. (2015) examined the response ofoil palm to defoliation stress. Obviously,understanding abiotic and biotic stress mechanismsis essential for the development of tolerant cropcultivars under the global climate change.ReferencesAjambang, W., S. W. Ardie, H. Volkaert, M.Galdima and S. Sudarsono. 2015. Hugecarbohydrate assimilates delay response tocomplete defoliation stress in oil palm (Elaeisguineensis Jacq.). Emir. J. Food Agric.27(1):126-137.Bakhsh, A. 2015. Genetic engineering of cropplants against abiotic stress: Current

Whole transcriptome profiling of maize during early somatic embryogenesis reveals altered expression of stress factors and embryogenesis-related genes.

Embryogenic tissue culture systems are utilized in propagation and genetic engineering of crop plants, but applications are limited by genotype-dependent culture response. To date, few genes necessary for embryogenic callus formation have been identified or characterized. The goal of this research was to enhance our understanding of gene expression during maize embryogenic tissue culture initiation. In this study, we highlight the expression of candidate genes that have been previously regarded in the literature as having important roles in somatic embryogenesis. We utilized RNA based sequencing (RNA-seq) to characterize the transcriptome of immature embryo explants of the highly embryogenic and regenerable maize genotype A188 at 0, 24, 36, 48, and 72 hours after placement of explants on tissue culture initiation medium. Genes annotated as functioning in stress response, such as glutathione-S-transferases and germin-like proteins, and genes involved with hormone transport, such as PINFORMED, increased in expression over 8-fold in the study. Maize genes with high sequence similarity to genes previously described in the initiation of embryogenic cultures, such as transcription factors BABY BOOM, LEAFY COTYLEDON, and AGAMOUS, and important receptor-like kinases such as SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASES and CLAVATA, were also expressed in this time course study. By combining results from whole genome transcriptome analysis with an in depth review of key genes that play a role in the onset of embryogenesis, we propose a model of coordinated expression of somatic embryogenesis-related genes, providing an improved understanding of genomic factors involved in the early steps of embryogenic culture initiation in maize and other plant species.

Role of Homeodomain Leucine Zipper (HD-Zip) IV Transcription Factors in Plant Development and Plant Protection from Deleterious Environmental Factors

Homeobox genes comprise an important group of genes that are responsible for regulation of developmental processes. These genes determine cell differentiation and cell fate in all eukaryotic organisms, starting from the early stages of embryo development. Homeodomain leucine zipper (HD-Zip) transcription factors are unique to the plant kingdom. Members of the HD-Zip IV subfamily have a complex domain topology and can bind several cis-elements with overlapping sequences. Many of the reported HD-Zip IV genes were shown to be specifically or preferentially expressed in plant epidermal or sub-epidermal cells. HD-Zip IV TFs were found to be associated with differentiation and maintenance of outer cell layers, and regulation of lipid biosynthesis and transport. Insights about the role of these proteins in plant cuticle formation, and hence their possible involvement in plant protection from pathogens and abiotic stresses has just started to emerge. These roles make HD-Zip IV proteins an attractive tool for genetic engineering of crop plants. To this end, there is a need for in-depth studies to further clarify the function of each HD-Zip IV subfamily member in commercially important plant species.

The Bsister MADS-box proteins have multiple regulatory functions in plant development

Abstract B sister MADS-box proteins are a group of recently characterized MADS family transcription factors. The genes encoding these proteins have a close relationship with the male-organ-expressed class B floral homeotic genes, but are predominantly expressed in female organs. B sister MADS-box proteins display important regulatory functions in seed coat development, fruit development, and proanthocyanidin biosynthesis. Therefore, genes encoding B sister MADS-box proteins are potentially useful in genetic engineering of crop plants to reduce seed coat thickness and condensed tannin content. In this review, recent progress on biochemical, molecular and functional aspects of plant B sister MADS-box proteins is discussed.

Genetic engineering of crop plants for fungal resistance: role of antifungal genes

Fungal diseases damage crop plants and affect agricultural production. Transgenic plants have been produced by inserting antifungal genes to confer resistance against fungal pathogens. Genes of fungal cell wall-degrading enzymes, such as chitinase and glucanase, are frequently used to produce fungal-resistant transgenic crop plants. In this review, we summarize the details of various transformation studies to develop fungal resistance in crop plants.

Characterization of Upstream Sequences of the LOJ Gene Leads to Identification of a Novel Enhancer Element Conferring Lateral Organ Junction-Specific Expression in Arabidopsis thaliana

Isolation and characterization of promoters are important in understanding gene regulation and genetic engineering of crop plants. Earlier, a pentatricopeptide repeat protein (PPR) encoding gene (At2g39230), designated as Lateral Organ Junction (LOJ) gene, was identified through T-DNA promoter trapping in Arabidopsis thaliana. The upstream sequence of the LOJ gene conferred on the reporter gene a novel LOJ-specific expression. The present study was aimed at identifying and characterizing the cis-regulatory motifs responsible for tissue-specific expression in the −673 and +90 bases upstream of the LOJ gene recognized as LOJ promoter. In silico analysis of the LOJ promoter revealed the presence of a few relevant regulatory motifs and a unique feature like AT-rich inverted repeat. Deletion analysis of the LOJ promoter confirmed the presence of an enhancer-like element in the distal region (−673/−214), which stimulates a minimal promoter-like sequence in the −424/−214 region in a position and orientation autonomous manner. The −136/+90 region of the LOJ promoter was efficient in driving reporter gene expression in tissues like developing anthers and seeds of Arabidopsis. A positive regulation for the seed- and anther-specific expression module was contemplated within the 5′ untranslated region of the LOJ gene. However, this function was repressed in the native context by the lateral organ junction-specific expression. The present study has led to the identification of a novel lateral organ junction-specific element and an enhancer sequence in Arabidopsis with potential applications in plant genetic engineering.

Rice endosperm iron biofortification by targeted and synergistic action of nicotianamine synthase and ferritin

Nearly 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 plasma-mass 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.