Enhancement of micronutrient bioavailability is crucial to address the malnutrition in the developing countries. Various approaches employed to address the micronutrient bioavailability are showing promising signs, especially in cereal crops. Phytic acid (PA) is considered as a major antinutrient due to its ability to chelate important micronutrients and thereby restricting their bioavailability. Therefore, manipulating PA biosynthesis pathway has largely been explored to overcome the pleiotropic effect in different crop species. Recently, we reported that functional wheat inositol pentakisphosphate kinase (TaIPK1) is involved in PA biosynthesis, however, the functional roles of the IPK1 gene in wheat remains elusive. In this study, RNAi-mediated gene silencing was performed for IPK1 transcripts in hexaploid wheat. Four non-segregating RNAi lines of wheat were selected for detailed study (S3-D-6-1; S6-K-3-3; S6-K-6-10 and S16-D-9-5). Homozygous transgenic RNAi lines at T4 seeds with a decreased transcript of TaIPK1 showed 28-56% reduction of the PA. Silencing of IPK1 also resulted in increased free phosphate in mature grains. Although, no phenotypic changes in the spike was observed but, lowering of grain PA resulted in the reduced number of seeds per spikelet. The lowering of grain PA was also accompanied by a significant increase in iron (Fe) and zinc (Zn) content, thereby enhancing their molar ratios (Zn:PA and Fe:PA). Overall, this work suggests that IPK1 is a promising candidate for employing genome editing tools to address the mineral accumulation in wheat grains.

/pdf/rnai-mediated-downregulation-of-inositol-pentakisphosphate-2oyvs2ifal.pdf

RNAi-Mediated Downregulation of Inositol Pentakisphosphate Kinase ( IPK1) in Wheat Grains Decreases Phytic Acid Levels and Increases Fe and Zn Accumulation

Zinc (Zn) deficiency is one of the most important micronutrient disorders affecting human health. Wheat is the staple food for 35% of the world’s population and is inherently low in Zn, which increases the incidence of Zn deficiency in humans. Major wheat-based cropping systems viz. rice–wheat, cotton–wheat and maize–wheat are prone to Zn deficiency due to the high Zn demand of these crops. This review highlights the role of Zn in plant biology and its effect on wheat-based cropping systems. Agronomic, breeding and molecular approaches to improve Zn nutrition and biofortification of wheat grain are discussed. Zinc is most often applied to crops through soil and foliar methods. The application of Zn through seed treatments has improved grain yield and grain Zn status in wheat. In cropping systems where legumes are cultivated in rotation with wheat, microorganisms can improve the available Zn pool in soil for the wheat crop. Breeding and molecular approaches have been used to develop wheat genotypes with high grain Zn density. Options for improving grain yield and grain Zn concentration in wheat include screening wheat genotypes for higher root Zn uptake and grain translocation efficiency, the inclusion of these Zn-efficient genotypes in breeding programs, and Zn fertilization through soil, foliar and seed treatments.

Zinc nutrition in wheat-based cropping systems

Zinc in plants: Integrating homeostasis and biofortification

Knowledge of genetic variation, genetics, physiology/molecular basis and breeding (including biotechnological approaches) for biofortification and bioavailability for Zn, Fe and Se will help in developing nutritionally improved wheat. Biofortification of wheat cultivars for micronutrients is a priority research area for wheat geneticists and breeders. It is known that during breeding of wheat cultivars for productivity and quality, a loss of grain micronutrient contents occurred, leading to decline in nutritional quality of wheat grain. Keeping this in view, major efforts have been made during the last two decades for achieving biofortification and bioavailability of wheat grain for micronutrients including Zn, Fe and Se. The studies conducted so far included evaluation of gene pools for contents of not only grain micronutrients as above, but also for phytic acid (PA) or phytate and phytase, so that, while breeding for the micronutrients, bioavailability is also improved. For this purpose, QTL interval mapping and GWAS were carried out to identify QTLs/genes and associated markers that were subsequently used for marker-assisted selection (MAS) during breeding for biofortification. Studies have also been conducted to understand the physiology and molecular basis of biofortification, which also allowed identification of genes for uptake, transport and storage of micronutrients. Transgenics using transgenes have also been produced. The breeding efforts led to the development of at least a dozen cultivars with improved contents of grain micronutrients, although land area occupied by these biofortified cultivars is still marginal. In this review, the available information on different aspects of biofortification and bioavailability of micronutrients including Zn, Fe and Se in wheat has been reviewed for the benefit of those, who plan to start work or already conducting research in this area.

Biofortification and bioavailability of Zn, Fe and Se in wheat: present status and future prospects.

Summary
The GT-1 cis-element widely exists in many plant gene promoters. However, the molecular mechanism that underlies the response of the GT-1 cis-element to abiotic and biotic stresses remains elusive in rice. We previously isolated a rice short-chain peptide-encoding gene, Os2H16, and demonstrated that it plays important roles in both disease resistance and drought tolerance. Here, we conducted a promoter assay of Os2H16 and identified GT-1 as an important cis-element that mediates Os2H16 expression in response to pathogen attack and osmotic stress. Using the repeated GT-1 as bait, we characterized an abscisic acid, stress and ripening 2 (ASR2) protein from yeast-one hybridization screening. Sequence alignments showed that the carboxy-terminal domain of OsASR2 containing residues 80–138 was the DNA-binding domain. Furthermore, we identified that OsASR2 was specifically bound to GT-1 and activated the expression of the target gene Os2H16, as well as GFP driven by the chimeric promoter of 2 × GT-1-35S mini construct. Additionally, the expression of OsASR2 was elevated by pathogens and osmotic stress challenges. Overexpression of OsASR2 enhanced the resistance against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani, and tolerance to drought in rice. These results suggest that the interaction between OsASR2 and GT-1 plays an important role in the crosstalk of the response of rice to biotic and abiotic stresses.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.12827

OsASR2 regulates the expression of a defence-related gene, Os2H16, by targeting the GT-1 cis-element.

Phytate is a major constituent of wheat seeds and chelates metal ions, thus reducing their bioavailability and so the nutritional value of grains. Transgenic plants expressing heterologous phytase are expected to enhance degradation of phytic acid stored in seeds and are proposed to increase the in vitro bioavailability of mineral nutrients. Wheat transgenic plants expressing Aspergillus japonicus phytase gene (phyA) in wheat endosperm were developed till T3 generation. The transgenic lines exhibited 18-99 % increase in phytase activity and 12-76 % reduction of phytic acid content in seeds. The minimum phytic acid content was observed in chapatti (Asian bread) as compared to flour and dough. The transcript profiling of phyA mRNA indicated twofold to ninefold higher expression as compared to non transgenic controls. There was no significant difference in grain nutrient composition of transgenic and non-transgenic seeds. In vitro bioavailability assay for iron and zinc in dough and chapatti of transgenic lines revealed a significant increase in iron and zinc contents. The development of nutritionally enhanced cereals is a step forward to combat nutrition deficiency for iron and zinc in malnourished human population, especially women and children.

Transgenic expression of phytase in wheat endosperm increases bioavailability of iron and zinc in grains

The regulatory sequence of sucrose synthase (susy) was explored in HTGS and screened using various bioinformatics tools for promoter prediction and identification of functional regulatory motifs. Transcription start site (TSS) was predicted in the promoter sequence. Species specific motifs were identified by using Plant PAN database. The Plant Care predicted various light responsive, hormone inducible and tissue specific motifs in the full length promoter which may be essential for the constitutive expression governed by this promoter. Full length susy promoter isolated from tomato (Solanum lycopersicum) drove maximum transient expression of GUS gene in various tissues of tobacco, cotton and peas. The susy promoter identified and analyzed in this study is suitable for transgene expression in economically important agricultural crops, especially to avoid strong over-expression.

Identification and analysis of an efficient dicot constitutive promoter from tomato

The outer epidermal layer of the cotton ovule may differentiate in vitro to form a mass of white fluffy fibers in an appropriate liquid medium. The in vitro cultured fibers provide a model for studying not only the cellular physiology and development mechanism but also generate a key resource for promoter evaluation in developing fibers. However, the potential uses of in vitro cultured ovules are restricted due to the frequent problem of bacterial and fungal contamination. In this study, the effect of Ampicillin, Gentamicin and Sodium Azide on cotton fiber development was observed at various concentrations (100 µg/mL, 50 µg/mL and 0.5 µg/mL respectively) which were added in the media as contamination controlling agents. The in vitro grown ovules were evaluated at 5, 10, 15 and 20 DPA (days post anthesis) for fiber length, percent viable ovules producing fiber, ovules fresh weight and ovules dry weight. It was observed that ovules grown on the media containing either Gentamicin or Sodium Azide were unable to initiate normal number of fiber cells. Addition of Ampicillin to cultures enhanced the fiber length, ovule weight and percentage of viable ovules with fibers. The inhibitory effect of Gentamicin on fiber growth was reversible, when ovules were transferred to Gentamicin -free medium within 3 days after culture initiation. However, Sodium Azide resulted in irreversible fiber development inhibition. These results indicate that Ampicillin was not only efficient to control contamination but also enhanced the fiber and ovule development process.

Effect of different antimicrobial agents on the fiber development of in vitro cultured cotton ovules

Identification, isolation and evaluation of a constitutive sucrose phosphate synthase gene promoter from tomato.

Cotton fiber genes and promoters are of great importance in understanding fiber development mechanism as well as for improvement of fiber. Sucrose phosphate synthase gene (SPS) (insert abbreviation of gene)has found to express at higher rate in developing cotton fibers. It is an important enzyme that have major role in sucrose as well as cellulose synthesis. Upstream region of a SPS gene from cotton was retrieved through HTGS database and analyzed using bioinformatics tools. Sequence analysis identified various regulatory motifs including light, drought, heat responsiveness and MYB binding sites in the promoter. The SPS promoter was isolated from cotton genomic DNA (using what method) and fused to β-glucuronidase (GUS) gene in a plant expression vector. Transient GUS expression analysis in various cotton tissues showed that promoter was active in the fiber tissues. Full length 2 kb SPS promoter showed high expression in fibers during elongation and secondary cell wall synthesis stage. A 1.5 kb deletion fragment showed reduced expression in fibers. Our results suggest that cotton SPS promoter may be used to express genes specifically in fiber cells for improvement of cotton fiber quality traits.

/pdf/isolation-and-characterization-of-sucrose-phosphate-synthase-58vv0dof2p.pdf

Isolation and characterization of sucrose phosphate synthase promoter from cotton (Gossypium hirsutum L.)

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