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Less than 10% of the estimated average requirement (EAR) for iron and zinc is provided by consumption of storage roots of the staple crop cassava (Manihot esculenta Crantz) in West African human populations. We used genetic engineering to improve mineral micronutrient concentrations in cassava. Overexpression of the Arabidopsis thaliana vacuolar iron transporter VIT1 in cassava accumulated three- to seven-times-higher levels of iron in transgenic storage roots than nontransgenic controls in confined field trials in Puerto Rico. Plants engineered to coexpress a mutated A. thaliana iron transporter (IRT1) and A. thaliana ferritin (FER1) accumulated iron levels 7–18 times higher and zinc levels 3–10 times higher than those in nontransgenic controls in the field. Growth parameters and storage-root yields were unaffected by transgenic fortification in our field data. Measures of retention and bioaccessibility of iron and zinc in processed transgenic cassava indicated that IRT1 + FER1 plants could provide 40–50% of the EAR for iron and 60–70% of the EAR for zinc in 1- to 6-year-old children and nonlactating, nonpregnant West African women.Cassava, a staple crop consumed by 800 million people, is enriched in iron and zinc through genetic biofortification.

Micronutrient deficiencies such as iron (Fe), zinc (Zn), and vitamin A, constitute a severe global public health phenomenon. Over half of preschool children and two-thirds of nonpregnant women of reproductive age worldwide have micronutrient deficiencies. Biofortification is a cost-effective strategy that comprises a meaningful and sustainable means of addressing this issue by delivering micronutrients through staple foods to populations with limited access to diverse diets and other nutritional interventions. Here, we report on the proof-of-concept and early development stage of a collection of biofortified rice events with a high density of Fe and Zn in polished grains that have been pursued further to advance development for product release. In total, eight constructs were developed specifically expressing dicot ferritins and the rice nicotianamine synthase 2 ( OsNAS2 ) gene under different combinations of promoters. A large-scale transformation of these constructs to Bangladesh and Philippines commercial indica cultivars and subsequent molecular screening and confined field evaluations resulted in the identification of a pool of ten events with Fe and Zn concentrations in polished grains of up to 11 μg g −1 and up to 37 μg g −1 , respectively. The latter has the potential to reduce the prevalence of inadequate Zn intake for women of childbearing age in Bangladesh and in the Philippines by 30% and 50%, respectively, compared to the current prevalence. To our knowledge, this is the first potential biotechnology public-sector product that adopts the product cycle phase-gated approach, routinely applied in the private sector.

The ability to control gene expression and generate quantitative phenotypic changes is essential for breeding new and desired traits into crops. Here we report an efficient, facile method for downregulating gene expression to predictable, desired levels by engineering upstream open reading frames (uORFs). We used base editing or prime editing to generate de novo uORFs or to extend existing uORFs by mutating their stop codons. By combining these approaches, we generated a suite of uORFs that incrementally downregulate the translation of primary open reading frames (pORFs) to 2.5–84.9% of the wild-type level. By editing the 5′ untranslated region of OsDLT , which encodes a member of the GRAS family and is involved in the brassinosteroid transduction pathway, we obtained, as predicted, a series of rice plants with varied plant heights and tiller numbers. These methods offer an efficient way to obtain genome-edited plants with graded expression of traits. Plant traits are fine-tuned by stepwise downregulation of gene expression.

Starch synthase III plays a key role in starch biosynthesis and is highly expressed in developing wheat grains. To understand the contribution of SSIII to starch and grain properties, we developed wheat ssIIIa mutants in the elite cultivar Cadenza using in silico TILLING in a mutagenized population. SSIIIa protein was undetectable by immunoblot analysis in triple ssIIIa mutants carrying mutations in each homoeologous copy of ssIIIa (A, B and D). Loss of SSIIIa in triple mutants led to significant changes in starch phenotype including smaller A-type granules and altered granule morphology. Starch chain-length distributions of double and triple mutants indicated greater levels of amylose than sibling controls (33.8% of starch in triple mutants, and 29.3% in double mutants vs. 25.5% in sibling controls) and fewer long amylopectin chains. Wholemeal flour of triple mutants had more resistant starch (6.0% vs. 2.9% in sibling controls) and greater levels of non-starch polysaccharides; the grains appeared shrunken and weighed ~ 11% less than the sibling control which was partially explained by loss in starch content. Interestingly, our study revealed gene dosage effects which could be useful for fine-tuning starch properties in wheat breeding applications while minimizing impact on grain weight and quality.

The conversion of lignocellulosic feedstocks to fermentable sugar for biofuel production is inefficient, and most strategies to enhance efficiency directly target lignin biosynthesis, with associated negative growth impacts. Here we demonstrate, for both laboratory- and field-grown plants, that expression of Pag-miR408 in poplar ( Populus alba × P. glandulosa ) significantly enhances saccharification, with no requirement for acid-pretreatment, while promoting plant growth. The overexpression plants show increased accessibility of cell walls to cellulase and scaffoldin cellulose-binding modules. Conversely, Pag-miR408 loss-of-function poplar shows decreased cell wall accessibility. Overexpression of Pag-miR408 targets three Pag-LACCASES , delays lignification, and modestly reduces lignin content, S/G ratio and degree of lignin polymerization. Meanwhile, the LACCASE loss of function mutants exhibit significantly increased growth and cell wall accessibility in xylem. Our study shows how Pag-miR408 regulates lignification and secondary growth, and suggest an effective approach towards enhancing biomass yield and saccharification efficiency in a major bioenergy crop. Modifying plant lignin pathway to enhance saccharification efficiency is often associated with growth penalty. Here, the authors show that overexpression of Pag-miR408 in poplar leads to enhanced saccharification efficiency and growth in both laboratory and field conditions, and laccase genes are the targets of Pag-miR408 .

Insufficient water availability for crop production is a mounting barrier to achieving the 70% increase in food production that will be needed by 2050. One solution is to develop crops that require less water per unit mass of production. Water vapor transpires from leaves through stomata, which also facilitate the influx of CO 2 during photosynthetic assimilation. Here, we hypothesize that Photosystem II Subunit S ( PsbS ) expression affects a chloroplast-derived signal for stomatal opening in response to light, which can be used to improve water-use efficiency. Transgenic tobacco plants with a range of PsbS expression, from undetectable to 3.7 times wild-type are generated. Plants with increased PsbS expression show less stomatal opening in response to light, resulting in a 25% reduction in water loss per CO 2 assimilated under field conditions. Since the role of PsbS is universal across higher plants, this manipulation should be effective across all crops. Availability of irrigation water will be an increasing barrier to global crop yield increases. Here the authors show transgenic tobacco plants overexpressing Photosystem II Subunit S have less stomatal opening in response to light and a 25% reduction in water loss per CO 2 assimilated under replicated field trials.

The insecticidal crystalline proteins (Crys) are a family of insect endotoxin functioning in crop protection. As insects keep evolving into tolerance to the existing Crys, it is necessary to discover new Cry proteins to overcome potential threatens. Crys possess three functional domains at their N-termini, and the most active region throughout evolution was found at the domain-III. We swapped domain-IIIs from various Cry proteins and generated seven chimeric proteins. All recombinants were expressed in Escherichia coli and their toxicity was assessed by dietary exposure assays. Three of the seven Crys exhibited a high toxicity to Asian corn borer over the controls. One of them, Cry1Ab-Gc, a chimeric Cry1Ab being replaced with the domain-III of Cry1Gc, showed the highest toxicity to rice stem borer when it was over-expressed in Oryza sativa . Furthermore, it was also transformed into maize, backcrossed into commercial maize inbred lines and then produced hybrid to evaluate their commercial value. Transgenic maize performed significant resistance to the Asian corn borer without affecting the yield. We further showed that this new protein did not have adverse effects on the environment. Our results indicated that domain III swapped of Crys could be used as an efficient method for developing new engineered insecticidal protein.

Engineered switchgrass and poplar are better feedstocks for biofuel synthesis and yield more biomass in multi-year field trials. Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene ( Galacturonosyltransferase 4 , GAUT4 ) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4 -knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Brazil is the world's largest producer of common bean. Knowledge of the genetic diversity and relatedness of accessions adapted to Brazilian conditions is of great importance for the conservation of germplasm and for directing breeding programs aimed at the development of new cultivars. In this context, the objective of this study was to analyze the genetic diversity, population structure, and linkage disequilibrium (LD) of a diversity panel consisting of 219 common bean accessions, most of which belonging to the Mesoamerican gene pool. Genotyping by sequencing (GBS) of these accessions allowed the identification of 49,817 SNPs with minor allele frequency > 0.05. Of these, 17,149 and 12,876 were exclusive to the Mesoamerican and Andean pools, respectively, and 11,805 SNPs could differentiate the two gene pools. Further the separation according to the gene pool, bayesian analysis of the population structure showed a subdivision of the Mesoamerican accessions based on the origin and color of the seed tegument. LD analysis revealed the occurrence of long linkage blocks and low LD decay with physical distance between SNPs (LD half decay in 249 kb, corrected for population structure and relatedness). The GBS technique could effectively characterize the Brazilian common bean germplasms, and the diversity panel used in this study may be of great use in future genome-wide association studies.

Tibetan wheat is grown under environmental constraints at high-altitude conditions, but its underlying adaptation mechanism remains unknown. Here, we present a draft genome sequence of a Tibetan semi-wild wheat ( Triticum aestivum ssp. tibetanum Shao) accession Zang1817 and re-sequence 245 wheat accessions, including world-wide wheat landraces, cultivars as well as Tibetan landraces. We demonstrate that high-altitude environments can trigger extensive reshaping of wheat genomes, and also uncover that Tibetan wheat accessions accumulate high-altitude adapted haplotypes of related genes in response to harsh environmental constraints. Moreover, we find that Tibetan semi-wild wheat is a feral form of Tibetan landrace, and identify two associated loci, including a 0.8-Mb deletion region containing Brt1/2 homologs and a genomic region with TaQ-5A gene, responsible for rachis brittleness during the de-domestication episode. Our study provides confident evidence to support the hypothesis that Tibetan semi-wild wheat is de-domesticated from local landraces, in response to high-altitude extremes. Mechanism of high altitude adaptation of wheat remains unknown. Here, the authors assemble the draft genome of a Tibetan semi-wild wheat accession and resequence 245 wheat accessions to reveal that Tibetan semi-wild wheat has been de-domesticated from local landraces to adapt to high altitude.