Search

Anthocyanins are important pigments for flower color, determining the ornamental and economic values of horticultural plants. As a key enzyme in the biosynthesis of anthocyanidins, dihydroflavonol 4-reductase (DFR) catalyzes the reduction of dihydroflavonols to generate the precursors for anthocyanidins (i.e., leucoanthocyanidins) and anthocyanins. To investigate the functions of DFRs in plants, we cloned the GlaDFR1 and GlaDFR2 genes from the petals of Gentiana lutea var. aurantiaca and transformed both genes into Nicotiana tabacum by Agrobacterium-mediated leaf disc method. We further investigated the molecular and phenotypic characteristics of T1 generation transgenic tobacco plants selected based on the hygromycin resistance and verified by both PCR and semiquantitative real-time PCR analyses. The phenotypic segregation was observed in the flower color of the transgenic tobacco plants, showing petals darker than those in the wild-type (WT) plants. Results of high-performance liquid chromatography (HPLC) analysis showed that the contents of gentiocyanin derivatives were decreased in the petals of transgenic plants in comparison to those of WT plants. Ours results revealed the molecular functions of GlaDFR1 and GlaDFR2 in the formation of coloration, providing solid theoretical foundation and candidate genes for further genetic improvement in flower color of plants.

To investigate the transrectal and scrotal ultrasonographic features of acquired obstructive azoospermia and evaluate the role of ultrasonography in the location diagnosis of acquired obstructive azoospermia patients.Retrospectively analysis of 92 cases of acquired obstructive azoospermia in recent years. All the patients underwent transrectal and scrotal ultrasonography. The ultrasonographic features were observed of testis, epididymis, scrotal segment of vas deferens, seminal vesicle, ejaculatory duct and prostate. Eighty cases with normal semen were taken as control group.Among the 92 cases of acquired obstructive azoospermia, 28 cases were prostate midline cyst, 32 cases were stone or calcification of the ejaculation duct and 21 cases were chronic seminal vesicle inflammation, which were found through transrectal ultrasonography; 27 cases were vas deferens dilation, 30 cases had abnormal mass of epididymis tail, 31 cases were epididymis thickness with reticular change, 8 cases were cystic dilatation of rete testis, which were found through scrotal ultrasonography. Compared with the control group, ultrasound examination increased the detection rate of distal seminal duct lesions and epididymal lesions in acquired obstructive azoospermia patients (p.01). Comparison of testicular volume between case group and control group did not reveal significant difference (p.05). The thickness of the head, body and tail of epididymis in case group was significantly bigger than that in control group (p.01).Transrectal and scrotal ultrasonography can find lesions in different parts of the seminal passage. Acquired obstructive azoospermia patients have increased rate of distal seminal duct lesions, epididymal lesions and epididymis volume. Transrectal and scrotum ultrasonography can provide reliable imaging evidence for the location diagnosis of acquired obstructive azoospermia.

Carotenoids are a large class of important lipid-soluble phytonutrients that are widely used as nutritional supplements due to their health-promoting activities. For example, β-carotene is the precursor for vitamin A synthesis, and astaxanthin is a powerful antioxidant. However, these carotenoids cannot be synthesized de novo by humans. These properties of β-carotene and astaxanthin make them attractive targets for metabolic engineering in rice (Oryza sativa) endosperm because rice is an important staple food in developing countries, and rice endosperm is devoid of carotenoids. In this chapter, we introduce an assay based on rice embryogenic callus for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation. The system is also an ideal platform to characterize cereal endosperm specific promoters. Four diverse cereal endosperm specific promoters were demonstrated to be active in rice callus despite their restricted activity in mature plants. The use of endosperm specific promoters that are expressed in rice callus, but remain silent in regenerated vegetative tissue, directs accumulation of carotenoids in the endosperm without interfering with plant growth. Rice callus is a useful platform for improving gene editing methods and for further optimizing pathway engineering. Thus, the rice callus platform provides a unique opportunity to test strategies for metabolic engineering of synthetic carotenoid pathways, leading to novel carotenoid-biofortified crops.

Plant biotechnology journal 19(10), 1921-1936 (2021). doi:10.1111/pbi.13658, Published by Wiley-Blackwell, Oxford

There are many reports indicating that biochar can promote growth; however, its mechanism of action remains unclear. The aim of this study was to show that organic molecules from biochar-extracted liquor affect the growth of rice seedlings. In this study, rice seedlings were cultured under water. Agronomic traits and growth-related genes and proteins were used as markers to describe more precisely the effects of biochar on specific growth parameters of rice seedlings. Our results demonstrated that the 3% biochar-extracted liquor amendment clearly promoted growth. The growth-related gene auxin binding protein 1 and its encoded protein were up-regulated. Molecular simulations revealed that 2-acetyl-5-methylfuran from biochar-extracted liquor could interact with auxin binding protein 1 in a similar way to indoleacetic acid binding. The growth of rice seedlings was therefore affected by biochar-extracted liquor, which acted on the ABP1 signalling pathway.

Anthocyanins are important pigments for flower color, determining the ornamental and economic values of horticultural plants. As a key enzyme in the biosynthesis of anthocyanidins, dihydroflavonol 4-reductase (DFR) catalyzes the reduction of dihydroflavonols to generate the precursors for anthocyanidins (i.e., leucoanthocyanidins) and anthocyanins. To investigate the functions of DFRs in plants, we cloned the GlaDFR1 and GlaDFR2 genes from the petals of Gentiana lutea var. aurantiaca and transformed both genes into Nicotiana tabacum by Agrobacterium-mediated leaf disc method. We further investigated the molecular and phenotypic characteristics of T1 generation transgenic tobacco plants selected based on the hygromycin resistance and verified by both PCR and semiquantitative real-time PCR analyses. The phenotypic segregation was observed in the flower color of the transgenic tobacco plants, showing petals darker than those in the wild-type (WT) plants. Results of high-performance liquid chromatography (HPLC) analysis showed that the contents of gentiocyanin derivatives were decreased in the petals of transgenic plants in comparison to those of WT plants. Ours results revealed the molecular functions of GlaDFR1 and GlaDFR2 in the formation of coloration, providing solid theoretical foundation and candidate genes for further genetic improvement in flower color of plants.

Isoprenoids are natural products derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, these precursors are synthesized via the cytosolic mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways. The regulation of these pathways must therefore be understood in detail to develop effective strategies for isoprenoid metabolic engineering. We hypothesized that the strict regulation of the native MVA pathway could be circumvented by expressing an ectopic plastidial MVA pathway that increases the accumulation of IPP and DMAPP in plastids. We therefore introduced genes encoding the plastid-targeted enzymes HMGS, tHMGR, MK, PMK and MVD and the nuclear-targeted transcription factor WR1 into rice and evaluated the impact of their endosperm-specific expression on (1) endogenous metabolism at the transcriptomic and metabolomic levels, (2) the synthesis of phytohormones, carbohydrates and fatty acids, and (3) the macroscopic phenotype including seed morphology. We found that the ectopic plastidial MVA pathway enhanced the expression of endogenous cytosolic MVA pathway genes while suppressing the native plastidial MEP pathway, increasing the production of certain sterols and tocopherols. Plants carrying the ectopic MVA pathway only survived if WR1 was also expressed to replenish the plastid acetyl-CoA pool. The transgenic plants produced higher levels of fatty acids, abscisic acid, gibberellins and lutein, reflecting crosstalk between phytohormones and secondary metabolism., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, project BIO2014-54426-P) and through the European Union Framework Program DISCO (from DISCOvery to products: a next-generation pipeline for the sustainable generation of high-value plant products, project 613513). LP was supported by a fellowship from MINECO

This meta-analysis aimed to evaluate the value of thyroid imaging report and data system (TI-RADS) combined with superb micro-vascular imagine technique(SMI) in distinguishing benign and malignant thyroid nodules. We searched PubMed, Web of Science, Cochrane Library, and Chinese biomedical databases from inception through February 31, 2021. Meta-analysis was conducted using STATA version 14.0 and Meta-Disc version 1.4 softwares. We calculated the summary statistics for sensitivity(Sen), specificity(Spe), and receiver operating characteristic (SROC) curve. Six studies that met all inclusion criteria were included in this meta-analysis. A total of 408 thyroid malignant nodules and 496 thyroid benign nodules were assessed. All thyroid nodules were histologically confirmed after SMI. The pooled Sen and Spe of TI-RADS were 0.80(95%CI = 0.71–0.87) and 0.82(95%CI = 0.75–0.87); The pooled Sen and Spe of TI-RADS combined with SMI were 0.88 (95%CI = 0.80–0.91) and 0.89 (95%CI = 0.85–0.92). The areas under the SROC curve of TI-RADS and TI-RADS combined with SMI were 0.8874(SE = 0.0165) and 0.9415(SE = 0.0102), between which there was significant difference(Z = 2.789; SE = 0.0194; p = 0.0053). Our meta-analysis indicates that TI-RADS combined with SMI may have high diagnostic accuracy, and is more effective than single TI-RADS in distinguishing benign and malignant thyroid nodules.

Multi-gene transformation methods need to be able to introduce multiple transgenes into plants in order to reconstitute a transgenic locus where the introduced genes express in a coordinated manner and do not segregate in subsequent generations. This simultaneous multiple gene transfer enables the study and modulation of the entire metabolic pathways and the elucidation of complex genetic control circuits and regulatory hierarchies. We used combinatorial nuclear transformation to produce multiplex-transgenic maize plants. In proof of principle experiments, we co-expressed five carotenogenic genes in maize endosperm. The resulting combinatorial transgenic maize plant population, equivalent to a "mutant series," allowed us to identify and complement rate-limiting steps in the extended endosperm carotenoid pathway and to recover corn plants with extraordinary levels of β-carotene and other nutritionally important carotenoids. We then introgressed the induced (transgenic) carotenoid pathway in a transgenic line accumulating high levels of nutritionally important carotenoids into a wild-type yellow-endosperm variety with a high β:ε ratio. Novel hybrids accumulated zeaxanthin at unprecedented amounts. We introgressed the same pathway into a different yellow corn line with a low β:ε ratio. The resulting hybrids, in this case, had a very different carotenoid profile. The role of genetic background in determining carotenoid profiles in corn was elucidated, and further rate-limiting steps in the pathway were identified and resolved in hybrids. Astaxanthin accumulation was engineered by overexpression of a β-carotene ketolase in maize endosperm. In early experiments, limited astaxanthin accumulation in transgenic maize plants was attributed to a bottleneck in the conversion of adonixanthin (4-ketozeaxanthin) to astaxanthin. More recent experiments showed that a synthetic β-carotene ketolase with a superior β-carotene/zeaxanthin ketolase activity is critical for the high-yield production of astaxanthin in maize endosperm. Engineered lines were used in animal feeding experiments which demonstrated not only the safety of the engineered lines but also their efficacy in a range of different animal production applications.

Genome-editing technologies offer unprecedented opportunities for crop improvement with superior precision and speed. This review presents an analysis of the current state of genome editing in the major cereal crops- rice, maize, wheat and barley. Genome editing has been used to achieve important agronomic and quality traits in cereals. These include adaptive traits to mitigate the effects of climate change, tolerance to biotic stresses, higher yields, more optimal plant architecture, improved grain quality and nutritional content, and safer products. Not all traits can be achieved through genome editing, and several technical and regulatory challenges need to be overcome for the technology to realize its full potential. Genome editing, however, has already revolutionized cereal crop improvement and is poised to shape future agricultural practices in conjunction with other breeding innovations. The authors would like to acknowledge funding from MINECO, Spain (PGC2018-097655-B-I00 to P Christou), Generalitat de Catalunya Grant 2017 SGR 828 to the Agricultural Biotechnology and Bioeconomy Unit (ABBU), Bill & Melinda Gates Foundation (to I. Slamet-Loedin).

Multi-gene transformation methods need to be able to introduce multiple transgenes into plants in order to reconstitute a transgenic locus where the introduced genes express in a coordinated manner and do not segregate in subsequent generations. This simultaneous multiple gene transfer enables the study and modulation of the entire metabolic pathways and the elucidation of complex genetic control circuits and regulatory hierarchies. We used combinatorial nuclear transformation to produce multiplex-transgenic maize plants. In proof of principle experiments, we co-expressed five carotenogenic genes in maize endosperm. The resulting combinatorial transgenic maize plant population, equivalent to a “mutant series,” allowed us to identify and complement rate-limiting steps in the extended endosperm carotenoid pathway and to recover corn plants with extraordinary levels of β-carotene and other nutritionally important carotenoids. We then introgressed the induced (transgenic) carotenoid pathway in a transgenic line accumulating high levels of nutritionally important carotenoids into a wild-type yellow-endosperm variety with a high β:e ratio. Novel hybrids accumulated zeaxanthin at unprecedented amounts. We introgressed the same pathway into a different yellow corn line with a low β:e ratio. The resulting hybrids, in this case, had a very different carotenoid profile. The role of genetic background in determining carotenoid profiles in corn was elucidated, and further rate-limiting steps in the pathway were identified and resolved in hybrids. Astaxanthin accumulation was engineered by overexpression of a β-carotene ketolase in maize endosperm. In early experiments, limited astaxanthin accumulation in transgenic maize plants was attributed to a bottleneck in the conversion of adonixanthin (4-ketozeaxanthin) to astaxanthin. More recent experiments showed that a synthetic β-carotene ketolase with a superior β-carotene/zeaxanthin ketolase activity is critical for the high-yield production of astaxanthin in maize endosperm. Engineered lines were used in animal feeding experiments which demonstrated not only the safety of the engineered lines but also their efficacy in a range of different animal production applications.

We summarize recent genome editing studies that have focused on the examination (or reexamination) of plant architectural phenotypes in cereals and the modification of these traits for crop improvement. Plant architecture is defined as the three-dimensional organization of the entire plant. Shoot architecture refers to the structure and organization of the aboveground components of a plant, reflecting the developmental patterning of stems, branches, leaves and inflorescences/flowers. Root system architecture is essentially determined by four major shape parameters—growth, branching, surface area and angle. Interest in plant architecture has arisen from the profound impact of many architectural traits on agronomic performance, and the genetic and hormonal regulation of these traits which makes them sensitive to both selective breeding and agronomic practices. This is particularly important in staple crops, and a large body of literature has, therefore, accumulated on the control of architectural phenotypes in cereals, particularly rice due to its twin role as one of the world’s most important food crops as well as a model organism in plant biology and biotechnology. These studies have revealed many of the molecular mechanisms involved in the regulation of tiller/axillary branching, stem height, leaf and flower development, root architecture and the grain characteristics that ultimately help to determine yield. The advent of genome editing has made it possible, for the first time, to introduce precise mutations into cereal crops to optimize their architecture and close in on the concept of the ideotype. In this review, we consider recent genome editing studies that have focused on the examination (or reexamination) of plant architectural phenotypes in cereals and the modification of these traits for crop improvement.

Starch properties can be modified by mutating genes responsible for the synthesis of amylose and amylopectin in the endosperm. However, little is known about the effects of such targeted modifications on the overall starch biosynthesis pathway and broader metabolism. Here we investigated the effects of mutating the OsSBEIIb gene encoding starch branching enzyme IIb, which is required for amylopectin synthesis in the endosperm. As anticipated, homozygous mutant plants, in which OsSBEIIb was completely inactivated by abolishing the catalytic center and C-terminal regulatory domain, produced opaque seeds with depleted starch reserves. Amylose content in the mutant increased from 19.6 to 27.4% and resistant starch (RS) content increased from 0.2 to 17.2%. Many genes encoding isoforms of AGPase, soluble starch synthase, and other starch branching enzymes were up-regulated, either in their native tissues or in an ectopic manner, whereas genes encoding granule-bound starch synthase, debranching enzymes, pullulanase, and starch phosphorylases were largely down-regulated. There was a general increase in the accumulation of sugars, fatty acids, amino acids, and phytosterols in the mutant endosperm, suggesting that intermediates in the starch biosynthesis pathway increased flux through spillover pathways causing a profound impact on the accumulation of multiple primary and secondary metabolites. Our results provide insights into the broader implications of perturbing starch metabolism in rice endosperm and its impact on the whole plant, which will make it easier to predict the effect of metabolic engineering in cereals for nutritional improvement or the production of valuable metabolites. We would like to acknowledge funding from Ministry of Economy and Competitiveness, Spain (RTI2018-097613-BI00 to C.Z., PGC2018-097655-B-I00 to P.C., and AGL2017-85377-R to T.C.); Generalitat de Catalunya Grant 2017 SGR 828 to the Agricultural Biotechnology and Bioeconomy Unit; and the European Union Framework Program DISCO (from discovery to final products: a next-generation pipeline for the sustainable generation of high-value plant products; Project 613513) to P.D.F.

Biofortification is an effective and economical method to improve the micronutrient content of crops, particularly staples that sustain human populations in developing countries. Whereas conventional fortification requires artificial additives, biofortification involves the synthesis or accumulation of nutrients by plants at source. Little is known about the relative merits of biofortification and artificial fortification in terms of nutrient bioaccessibility and bioavailability, and much depends on the biochemical nature of the nutrient, which can promote or delay uptake, and determine how efficiently different nutrients are transported through the blood, stored, and utilized. Data from the first plants biofortified with minerals and vitamins provide evidence that the way in which nutrients are presented can affect how they are processed and utilized in the human body. The latest studies on the effects of the food matrix, processing and storage on nutrient transfer from biofortified crops are reviewed, as well as current knowledge about nutrient absorption and utilization. Research at the University of Lleida is supported by MINECO, Spain (BIO2014-54426-P; BIO2014-54441-P, FEDER funds), the Catalan Government (2014 SGR 1296 Agricultural Biotechnology Research Group) and Recercaixa. J. Díaz-Gómez thanks the University of Lleida for a pre-doctoral grant.

Mitochondria fulfil essential functions in respiration and metabolism as well as regulating stress responses and apoptosis. Most native mitochondrial proteins are encoded by nuclear genes and are imported into mitochondria via one of several receptors that recognize N-terminal signal peptides. The targeting of recombinant proteins to mitochondria therefore requires the presence of an appropriate N-terminal peptide, but little is known about mitochondrial import in monocotyledonous plants such as rice (Oryza sativa). To gain insight into this phenomenon, we targeted nuclear-encoded enhanced green fluorescent protein (eGFP) to rice mitochondria using six mitochondrial pre-sequences with diverse phylogenetic origins, and investigated their effectiveness by immunoblot analysis as well as confocal and electron microscopy. We found that the ATPA and COX4 (Saccharomyces cerevisiae), SU9 (Neurospora crassa), pFA (Arabidopsis thaliana) and OsSCSb (Oryza sativa) peptides successfully directed most of the eGFP to the mitochondria, whereas the MTS2 peptide (Nicotiana plumbaginifolia) showed little or no evidence of targeting ability even though it is a native plant sequence. Our data therefore indicate that the presence of particular recognition motifs may be required for mitochondrial targeting, whereas the phylogenetic origin of the pre-sequences probably does not play a key role in the success of mitochondrial targeting in dedifferentiated rice callus and plants.

Both OsIPPI1 and OsIPPI2 enzymes are found in the endoplasmic reticulum, providing novel important insights into the role of this compartment in the synthesis of MVA pathway isoprenoids. Isoprenoids are synthesized from the precursor’s isopentenyl diphosphate (IPP) and dimethylallyl diphosphosphate (DMAPP), which are interconverted by the enzyme isopentenyl diphosphate isomerase (IPPI). Many plants express multiple isoforms of IPPI, the only enzyme shared by the mevalonate (MVA) and non-mevalonate (MEP) pathways, but little is known about their specific roles. Rice (Oryza sativa) has two IPPI isoforms (OsIPPI1 and OsIPPI2). We, therefore, carried out a comprehensive comparison of IPPI gene expression, protein localization, and isoprenoid biosynthesis in this species. We found that OsIPPI1 mRNA was more abundant than OsIPPI2 mRNA in all tissues, and its expression in de-etiolated leaves mirrored the accumulation of phytosterols, suggesting a key role in the synthesis of MVA pathway isoprenoids. We investigated the subcellular localization of both isoforms by constitutively expressing them as fusions with synthetic green fluorescent protein. Both proteins localized to the endoplasmic reticulum (ER) as well as peroxisomes and mitochondria, whereas only OsIPPI2 was detected in plastids, due to an N-terminal transit peptide which is not present in OsIPPI1. Despite the plastidial location of OsIPPI2, the expression of OsIPPI2 mRNA did not mirror the accumulation of chlorophylls or carotenoids, indicating that OsIPPI2 may be a redundant component of the MEP pathway. The detection of both OsIPPI isoforms in the ER indicates that DMAPP can be synthesized de novo in this compartment. Our work shows that the ER plays an as yet unknown role in the synthesis of MVA-derived isoprenoids, with important implications for the metabolic engineering of isoprenoid biosynthesis in higher plants.

Maize is a staple food crop in many developing countries, hence becoming an attractive target for biofortification programs toward populations at risk of micronutrient deficiencies. A South African white endosperm maize inbred line was engineered with a carotenogenic mini-pathway to generate high-carotenoid maize, which accumulates β-carotene, lutein and zeaxanthin. As maize porridge is a traditional meal for poor populations in sub-Saharan African countries, high-carotenoid maize was used as raw material to prepare different maize meals. The objective of this work was to assess the impact of popular home-cooking techniques and different cooking parameters (temperature, time and pH) on the final carotenoid content in the cooked product, using a spectrophotometric technique based on the mean absorption of carotenoids at 450 nm. Carotenoid levels were not only preserved, but also enhanced in high-carotenoid maize porridges. The carotenoid content was increased when temperatures ≤95 °C were combined with short cooking times (10-60 min). The most optimum thermal treatment was 75 °C/10 min. When treated under those conditions at pH 5, high-carotenoid maize porridges doubled the initial carotenoid content up to 88 μg/g dry weight. Regarding to cooking techniques, the highest carotenoid content was found when unfermented thin porridges were prepared (51 μg/g dry weight of high-carotenoid maize porridge). We conclude that high-carotenoid maize may contribute to enhance the dietary status of rural populations who depend on maize as a staple food.

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.

Astaxanthin from a transgenic maize line was evaluated as feed supplement source conferring effective pigmentation of rainbow trout flesh. An extraction procedure using ethanol together with the addition of vegetal oil was established. This resulted in an oily astaxanthin preparation which was not sufficiently concentrated for direct application to the feed. Therefore, a concentration process involving multiple phase partitioning steps was implemented to remove 90 % of the oil. The resulting astaxanthin raw material contained non-esterified astaxanthin with 12 % 4-keto zeaxanthin and 2 % zeaxanthin as additional carotenoids. Isomeric analysis confirmed the exclusive presence of the 3S, 3'S astaxanthin enantiomer. The geometrical isomers were 89 % all-E, 8 % 13-Z and 3 % 9-Z. The incorporation of the oily astaxanthin preparation into trout feed was performed to deliver 7 mg/kg astaxanthin in the final feed formulation for the first 3.5 weeks and 72 mg/kg for the final 3.5 weeks of the feeding trial. The resulting pigmentation of the trout fillets was determined by hue values with a colour meter and further confirmed by astaxanthin quantification. Pigmentation properties of the maize-produced natural astaxanthin incorporated to 3.5 µg/g dw in the trout fillet resembles that of chemically synthesized astaxanthin. By comparing the relative carotenoid compositions in feed, flesh and feces, a preferential uptake of zeaxanthin and 4-keto zeaxanthin over astaxanthin was observed.

Main conclusion The ratio of nicotianamine to deoxymugenic acid controls tissue-specific metal homeostasis in rice and regulates metal delivery to the endosperm. Abstract The metal-chelating phytosiderophores nicotianamine (NA) and 2′deoxymugenic acid (DMA) are significant factors for the control of metal homeostasis in graminaceous plants. These compounds are thought to influence metal homeostasis, but their individual roles and the effect of altering the NA:DMA ratio are unknown. We purposely generated rice lines with high and low NA:DMA ratios (HND and LND lines, respectively). The HND lines accumulated more iron (Fe), zinc (Zn), manganese (Mn) and copper (Cu) in the endosperm through the mobilization of Fe, Zn and Mn from the seed husk to the endosperm. In contrast, Fe, Zn and Mn were mobilized to the husk in the LND lines, whereas Cu accumulated in the endosperm. Different groups of metals are, therefore, taken up, transported and sequestered in vegetative tissues in the HND and LND lines to achieve this metal distribution pattern in the seeds. We found that different sets of endogenous metal homeostasis genes were modulated in the HND and LND lines to achieve differences in metal homeostasis. Our findings demonstrate that the NA:DMA ratio is a key factor regulating metal homeostasis in graminaceous plants. These findings can help formulate refined strategies to improve nutrient composition and nutrient use efficiency in crop plants., We acknowledge support from the European Research Council IDEAS Advanced Grant Program (BIOFORCE) to P.C., Generalitat de Catalunya Grant 2017 SGR 828 to ABBU (BIO2014-54426-P) and the Spanish Ministry of Economy and Competitivity (MINECO; projects AGL2016-75226-R, co-financed with FEDER) and the Aragón Government (Group A09_17R) to J.A. R.B was supported by a PhD fellowship from the University of Lleida, Spain.

Multiplex genome editing involves the simultaneous targeting of multiple related or unrelated targets. The latter is most straightforward using the CRISPR/Cas9 system because multiple gRNAs can be delivered either as independent expression cassettes with their own promoters or as polycistronic transcripts processed into mature gRNAs by endogenous or introduced nucleases. Multiplex genome editing in plants initially focused on input traits such as herbicide resistance, but has recently expanded to include hormone biosynthesis and perception, metabolic engineering, plant development and molecular farming, with more than 100 simultaneous targeting events reported. Usually the coding region is targeted but recent examples also include promoter modifications to generate mutants with varying levels of gene expression.

The first committed step in the endosperm starch biosynthetic pathway is catalyzed by the cytosolic glucose-1-phosphate adenylyl transferase (AGPase) comprising large and small subunits encoded by the OsAPL2 and OsAPS2b genes, respectively. OsAPL2 is expressed solely in the endosperm so we hypothesized that mutating this gene would block starch biosynthesis in the endosperm without affecting the leaves. We used CRISPR/Cas9 to create two heterozygous mutants, one with a severely truncated and nonfunctional AGPase and the other with a C-terminal structural modification causing a partial loss of activity. Unexpectedly, we observed starch depletion in the leaves of both mutants and a corresponding increase in the level of soluble sugars. This reflected the unanticipated expression of both OsAPL2 and OsAPS2b in the leaves, generating a complete ectopic AGPase in the leaf cytosol, and a corresponding decrease in the expression of the plastidial small subunit OsAPS2a that was only partially complemented by an increase in the expression of OsAPS1. The new cytosolic AGPase was not sufficient to compensate for the loss of plastidial AGPase, most likely because there is no wider starch biosynthesis pathway in the leaf cytosol and because pathway intermediates are not shuttled between the two compartments.

The apocarotenoid crocetin and its glycosylated derivatives, crocins, confer the red colour to saffron. Crocetin biosynthesis in saffron is catalysed by the carotenoid cleavage dioxygenase CCD2 (AIG94929). No homologues have been identified in other plant species due to the very limited presence of crocetin and its derivatives in the plant kingdom. Spring Crocus species with yellow flowers accumulate crocins in the stigma and tepals. Four carotenoid CCDs, namely CaCCD1, CaCCD2 and CaCCD4a/b and CaCCD4c were first cloned and characterized. CaCCD2 was localized in plastids, and a longer CCD2 version, CsCCD2L, was also localized in this compartment. The activity of CaCCD2 was assessed in Escherichia coli and in a stable rice gene function characterization system, demonstrating the production of crocetin in both systems. The expression of all isolated CCDs was evaluated in stigma and tepals at three key developmental stages in relation with apocarotenoid accumulation. CaCCD2 expression parallels crocin accumulation, but C14 apocarotenoids most likely are associated to the CaCCD1 activity in Crocus ancyrensis flowers. The specific CCD2 localization and its membrane interaction will contribute to the development of a better understanding of the mechanism of crocetin biosynthesis and regulation in the chromoplast.

The profile of secondary metabolites in plants reflects the balance of biosynthesis, degradation and storage, including the availability of precursors and products that affect the metabolic equilibrium. We investigated the impact of the precursor–product balance on the carotenoid pathway in the endosperm of intact rice plants because this tissue does not normally accumulate carotenoids, allowing us to control each component of the pathway. We generated transgenic plants expressing the maize phytoene synthase gene (ZmPSY1) and the bacterial phytoene desaturase gene (PaCRTI), which are sufficient to produce β-carotene in the presence of endogenous lycopene β-cyclase. We combined this mini-pathway with the Arabidopsis thaliana genes AtDXS (encoding 1-deoxy-D-xylulose 5-phosphate synthase, which supplies metabolic precursors) or AtOR (the ORANGE gene, which promotes the formation of a metabolic sink). Analysis of the resulting transgenic plants suggested that the supply of isoprenoid precursors from the MEP pathway is one of the key factors limiting carotenoid accumulation in the endosperm and that the overexpression of AtOR increased the accumulation of carotenoids in part by up-regulating a series of endogenous carotenogenic genes. The identification of metabolic bottlenecks in the pathway will help to refine strategies for the creation of engineered plants with specific carotenoid profiles. This study was supported by the Ministerio de Economia y Competitividad, Spain (BIO2011-22525 and PIM2010PKB-00746 CAROMAIZE), European Research Council Advanced Grant (BIOFORCE) to PC. C.B. is the recipient of a PhD fellowship from the Universitat de Lleida, Spain.

Carotenoids are lipophilic isoprenoid compounds synthesized by all photosynthetic organisms and some non-photosynthetic prokaryotes and fungi. With some notable exceptions, animals (including humans) do not produce carotenoids de novo but take them in their diets. In photosynthetic systems carotenoids are essential for photoprotection against excess light and contribute to light harvesting, but perhaps they are best known for their properties as natural pigments in the yellow to red range. Carotenoids can be associated to fatty acids, sugars, proteins, or other compounds that can change their physical and chemical properties and influence their biological roles. Furthermore, oxidative cleavage of carotenoids produces smaller molecules such as apocarotenoids, some of which are important pigments and volatile (aroma) compounds. Enzymatic breakage of carotenoids can also produce biologically active molecules in both plants (hormones, retrograde signals) and animals (retinoids). Both carotenoids and their enzymatic cleavage products are associated with other processes positively impacting human health. Carotenoids are widely used in the industry as food ingredients, feed additives, and supplements. This review, contributed by scientists of complementary disciplines related to carotenoid research, covers recent advances and provides a perspective on future directions on the subjects of carotenoid metabolism, biotechnology, and nutritional and health benefits., Authors belong to the Spanish Carotenoid Network (CaRed) funded by the Spanish MINECO (Ministry of Economy, Industry and Competitiveness) grant BIO2015-71703-REDT, the European Carotenoid Network (EuroCaroten) funded by the European Commission COST ActionCA15136, and the Ibero-American Carotenoid Network (IberCarot) funded by the Ibero-American Programme for Science, Technology and Development (CYTED) grant 112RT0445. Research on carotenoids at the authors' individual laboratories was supported by Spanish MINECO grants BIO2014-59092-P to MRC, BIO2015-69613-R to JA and MCL, AGL2013-43522-R to AB, BIO2016-77000-R to LGG, AGL2014-53195-R to DHM, AGL2015-67019-P to AP, BIO2014-54441-P to CZ, and AGL2015-70218 to LZ and MJR; Instituto de Salud Carlos III-FEDER grant PI16/01991 to BOA; Generalitat de Catalunya grants 214SGR-1434 to MRC and AB and 2014SGR-1296 to CZ; and Junta de Andalucia grants P10-CTS-6638 to JA and P12-AGR-1287 to AJMM. We also acknowledge the financial support of the CERCA Programme of the Generalitat de Catalunya and the Severo Ochoa Programme for Centres of Excellence in R&D 2016–2019 to CRAG (SEV-2015-0533).

Maize (Zea mays L.) is a staple food in many parts of Africa, but the endosperm generally contains low levels of the pro-vitamin A carotenoid -carotene, leading to vitamin A deficiency disease in populations relying on cereal-based diets. However, maize endosperm does accumulate high levels of other carotenoids, including zeaxanthin, which is derived from -carotene via two hydroxylation reactions. Blocking these reactions could therefore improve the endosperm -carotene content. Accordingly, we used RNA interference (RNAi) to silence the endogenous ZmBCH1 and ZmBCH2 genes, which encode two non-heme di-iron carotenoid -hydroxylases. The genes were silenced in a range of maize genetic backgrounds by introgressing the RNAi cassette, allowing us to determine the impact of ZmBCH1/ZmBCH2 silencing in diverse hybrids. The -carotene content of the endosperm increased substantially in all hybrids in which ZmBCH2 was silenced, regardless of whether or not ZmBCH1 was silenced simultaneously. However, the -carotene content did not change significantly in C17 hybrids (M7 C17 and M13 C17) compared to C17 alone, because ZmBCH2 is already expressed at negligible levels in the C17 parent. Our data indicate that ZmBCH2 is primarily responsible for the conversion of -carotene to zeaxanthin in maize endosperm

Carolight® is a transgenic maize variety that accumulates extraordinary levels of carotenoids, including those with vitamin A activity. The development of Carolight® maize involved the technical implementation of a novel combinatorial transformation method, followed by rigorous testing for transgene expression and the accumulation of different carotenoid molecules. Carolight® was envisaged as a way to improve the nutritional health of human populations that cannot access a diverse diet, but this ultimate humanitarian application can only be achieved after extensive testing for safety, agronomic performance and nutritional sufficiency. In this article, we chart the history of Carolight® maize focusing on its development, extensive field testing for agronomic performance and resistance to pests and pathogens, and feeding trials to analyze its impact on farm animals (and their meat/dairy products) as well as animal models of human diseases. We also describe more advanced versions of Carolight® endowed with pest-resistance traits, and other carotenoid-enhanced maize varieties originating from the same series of initial transformation experiments. Finally we discuss the further steps required before Carolight® can fulfil its humanitarian objectives, including the intellectual property and regulatory constraints that lie in its path. European Research Council Advanced Grant (BIOFORCE) and Proof of Concept Grant (Multinutrient Maize) to PC; Spanish Ministry of Economy and Competitiveness (AGL2014-53970-C2-1-R, AGL2015-65846-R, BIO2014-54426-P and BIO2014-54441-P); RecerCaixa project PC084082; Catalan Autonomous Community 2014 SGR 1296 Agricultural Biotechnology Research Group and Agrotecnio Research Center. JDG thanks the UdL for a Ph.D. fellowship.

Carotenoids are nutritionally-beneficial organic tetraterpenoid pigments synthesized mainly by plants, bacteria and fungi. Although research has focused on the production of carotenoids in staple crops to improve nutritional welfare in developing countries, there is also an enormous market for carotenoids in the industrialized world, where they are produced both as commodities and luxury goods targeted at the pharmaceutical, nutraceutical, food/feed additive, cosmetics and fine chemicals sectors. Carotenoids are economically valuable because they have diverse bioactive and chemical properties. Some are essential nutrients (e.g. β-carotene), others are antioxidants with specific roles (e.g. lutein and zeaxanthin) or general health-promoting roles that reduce the risk or progression of diseases associated with oxidative stress (e.g. lycopene), and still others are natural pigments (e.g. astaxanthin, which is added to fish feed to impart a desirable pink flesh color). Even carotenoid degradation products, such as damascones and damascenones, are used as fragrances in the perfumes industry. Here we discuss the importance of carotenoids in different market sectors, review current methods for commercial production and its regulation, summarize the most relevant patents and consider evidence supporting the health claims made by different industry sectors, focusing on case studies representing the most commercially valuable carotenoids on the market: β-carotene, lycopene, lutein, zeaxanthin and astaxanthin.

Rice endosperm is devoid of carotenoids because the initial biosynthetic steps are absent. The early carotenogenesis reactions were constituted through co-transformation of endosperm-derived rice callus with phytoene synthase and phytoene desaturase transgenes. Subsequent steps in the pathway such as cyclization and hydroxylation reactions were catalyzed by endogenous rice enzymes in the endosperm. The carotenoid pathway was extended further by including a bacterial ketolase gene able to form astaxanthin, a high value carotenoid which is not a typical plant carotenoid. In addition to astaxanthin and precursors, a carotenoid accumulated in the transgenic callus which did not fit into the pathway to astaxanthin. This was subsequently identified as 4-keto-a-carotene by HPLC co-chromatography, chemical modification, mass spectrometry and the reconstruction of its biosynthesis pathway in Escherichia coli. We postulate that this keto carotenoid is formed from a-carotene which accumulates by combined reactions of the heterologous gene products and endogenous rice endosperm cyclization reactions. This study was funded by the European Union Framework 7 European Research Council IDEAS Advanced Grant (to PC) Program-BIOFORCE, RecerCaixa, Ministerio de Ciencia e Innovación, Spain (BIO02011-22525; PIM2010PKB-00746) and Plant KBBE CaroMaize project.

We have developed an assay based on rice embryogenic callus for rapid functional characterization of metabolic genes. We validated the assay using a selection of well-characterized genes with known functions in the carotenoid biosynthesis pathway, allowing rapid visual screening of callus phenotypes based on tissue color. We then used the system to identify the functions of two uncharacterized genes: a chemically synthesized β–carotene ketolase gene optimized for maize codon usage, and a wild-type Arabidopsis thaliana ortholog of the cauliflower Orange gene. In contrast to previous reports (Lopez, A.B., Van Eck, J., Conlin, B.J., Paolillo, D.J., O'Neill, J. and Li, L. (2008) J. Exp. Bot. 59, 213–223; Lu, S., Van Eck, J., Zhou, X., Lopez, A.B., O'Halloran, D.M., Cosman, K.M., Conlin, B.J., Paolillo, D.J., Garvin, D.F., Vrebalov, J., Kochian, L.V., Küpper, H., Earle, E.D., Cao, J. and Li, L. (2006) Plant Cell 18, 3594–3605), we found that the wild-type Orange allele was sufficient to induce chromoplast differentiation. We also found that chromoplast differentiation was induced by increasing the availability of precursors and thus driving flux through the pathway, even in the absence of Orange. Remarkably, we found that diverse endosperm-specific promoters were highly active in rice callus despite their restricted activity in mature plants. Our callus system provides a unique opportunity to predict the effect of metabolic engineering in complex pathways, and provides a starting point for quantitative modeling and the rational design of engineering strategies using synthetic biology. We discuss the impact of our data on analysis and engineering of the carotenoid biosynthesis pathway. This study was supported by the Ministerio de Ciencia e Innovaci _on, Spain (AGL2010-15691, BFU2010-17704 and BIO2011-22525), the Generalitat de Catalunya (2009SGR809), and a European Research Council Advanced Grant (BIOFORCE). We thank Veronica Teixido and Jorge Comas (Department Cie` ncies Me` diques Ba` siques, Universitat de Lleida & Institut de Recerca Biomèdica de Lleida Edifici Recerca Biomèdica I, Avenida Alcalde Rovira Roure 80, 25198 Lleida, Spain) for assistance with the ImageJ analysis.

Maize (Zea mays spp. mays) is a staple crop for more than 900 million people. The seeds or kernels provide a rich source of calories because ~70 % of the weight is carbohydrate, mostly in the form of starch. The content and composition of starch are complex traits controlled by many genes, offering multiple potential targets for intervention. We used a multigene engineering approach combining the overexpression of Bt2, Sh2, Sh1 and GbssIIa (to enhance the activity of sucrose synthase, AGPase and granule-bound starch synthase) with the suppression of SbeI and SbeIIb by RNA interference (to reduce the activity of starch branching enzyme). Maize plants expressing all six genes plus the selectable marker showed a 2.8–7.7 % increase in the endosperm starch content and a 37.8–43.7 % increase in the proportion of amylose, which was significant compared to untransformed control plants. We also observed improvements in other agronomic traits, such as a 20.1–34.7 % increase in 100-grain weight, a 13.9–19.0 % increase in ear weight, and larger kernels with a better appearance, presumably reflecting the modified starch structure within the kernels. Our results confirm that multigene engineering applied to the starch biosynthesis pathway can not only modulate the quality and quantity of starch but can also improve starch-dependent agronomic traits.

Carotenoids are a diverse group of tetraterpenoid pigments found in plants, fungi, bacteria and some animals. They play vital roles in plants and provide important health benefits to mammals, including humans. We previously reported the creation of a diverse population of transgenic maize plants expressing various carotenogenic gene combinations and exhibiting distinct metabolic phenotypes. Here we performed an in-depth targeted mRNA and metabolomic analysis of the pathway to characterize the specific impact of five carotenogenic transgenes and their interactions with 12 endogenous genes in four transgenic lines representing distinct genotypes and phenotypes. We reconstructed the temporal profile of the carotenoid pathway during endosperm development at the mRNA and metabolic levels (for total and individual carotenoids), and investigated the impact of transgene expression on the endogenous pathway. These studies enabled us to investigate the extent of any interactions between the introduced transgenic and native partial carotenoid pathways during maize endosperm development. Importantly, we developed a theoretical model that explains these interactions, and our results suggest genetic intervention points that may allow the maize endosperm carotenoid pathway to be engineered in a more effective and predictable manner. This study was supported by the Ministerio de Ciencia e Innovación, Spain (BIO2007-61413, BIO2011-22525 and PM2010PKB-00746 CAROMAIZE) and a European Research Council Advanced Grant (BIOFORCE) to P.C. G.F. is the recipient of a PhD fellowship from the Ministerio de Ciencia e Innovacion, Spain (BES-2008-003894). R.A. and A.S. are partially funded through Research Group 2009SGR809 from the Generalitat de Catalunya and through grants BFU2008-0196 to A.S. and BFU2010-17704 to R.A., both from the Ministerio de Ciencia e Innovacion, Spain.

The AtOR gene enhances carotenoid levels in corn by promoting the formation of plastoglobuli when the carotenoid pool is limited, but has no further effect when carotenoids are already abundant. The cauliflower orange (or) gene mutation influences carotenoid accumulation in plants by promoting the transition of proplastids into chromoplasts, thus creating intracellular storage compartments that act as metabolic sink. We overexpressed the Arabidopsis OR gene under the control of the endosperm-specific wheat LMW glutenin promoter in a white corn variety that normally accumulates only trace amounts of carotenoids. The total endosperm carotenoid content in the best-performing AtOR transgenic corn line was 32-fold higher than wild-type controls (~25 µg/g DW at 30 days after pollination) but the principal carotenoids remained the same, suggesting that AtOR increases the abundance of existing carotenoids without changing the metabolic composition. We analyzed the expression of endogenous genes representing the carotenoid biosynthesis and MEP pathways, as well as the plastid fusion/translocation factor required for chromoplast formation, but only the DXS1 gene was upregulated in the transgenic corn plants. The line expressing AtOR at the highest level was crossed with four transgenic corn lines expressing different carotenogenic genes and accumulating different carotenoids. The introgression of AtOR increased the carotenoid content of the hybrids when there was a limited carotenoid pool in the parental line, but had no effect when carotenoids were already abundant in the parent. The AtOR gene therefore appears to enhance carotenoid levels by promoting the formation of carotenoid-sequestering plastoglobuli when the carotenoid pool is limited, but has no further effect when carotenoids are already abundant because high levels of carotenoids can induce the formation of carotenoid-sequestering plastoglobuli even in the absence of AtOR.

High-carotenoid (HC) maize, a biofortified staple crop which accumulates β-carotene, β-cryptoxanthin, lutein and zeaxanthin, was used as a feed component in a chicken feeding trial to assess the bioavailability of provitamin A (PVA) carotenoids in the kernel matrix compared to the synthetic and natural color additives routinely used in the poultry industry. We found that the PVA carotenoids in HC maize were not metabolized in the same manner: β-carotene was preferentially converted into retinol in the intestine whereas β-cryptoxanthin accumulated in the liver. We also considered the effect of zeaxanthin on the absorption of PVA carotenoids because zeaxanthin is the major carotenoid component of HC maize. We found that chickens fed on diets with low levels of zeaxanthin accumulated higher levels of retinol in the liver, suggesting that zeaxanthin might interfere with the absorption of β-carotene, although this observation was not statistically significant. Our results show that HC maize provides bioavailable carotenoids, including PVA carotenoids, and is suitable for use as a feed component. This work was supported by La Caixa (Recercaixa project PC084082 VitaMaize: High quality and safe food through antioxidant fortified maize), the Spanish Ministry of Economy and Competitiveness (BIO2014-54426-P; BIO2014-54441-P, FEDER funds), the Catalan Government (2014 SGR 1296 Agricultural Biotechnology Research Group) and the Agrotecnio Center.

Flower color is an important characteristic that determines the commercial value of ornamental plants. Gentian flowers occur in a limited range of colors because this species is not widely cultivated as a cut flower. Gentiana lutea L. var. aurantiaca (abbr, aurantiaca) is characterized by its orange flowers, but the specific pigments responsible for this coloration are unknown. We therefore investigated the carotenoid and flavonoid composition of petals during flower development in the orange-flowered gentian variety of aurantiaca and the yellow-flowered variety of G. lutea L. var. lutea (abbr, lutea). We observed minor varietal differences in the concentration of carotenoids at the early and final stages, but only aurantiaca petals accumulated pelargonidin glycosides, whereas these compounds were not found in lutea petals. We cloned and sequenced the anthocyanin biosynthetic gene fragments from petals, and analyzed the expression of these genes in the petals of both varieties to determine the molecular mechanisms responsible for the differences in petal color. Comparisons of deduced amino acid sequences encoded by the isolated anthocyanin cDNA fragments indicated that chalcone synthase (CHS), chalcone isomerase (CHI), anthocyanidin synthase 1 (ANS1) and ANS2 are identical in both aurantiaca and lutea varieties whereas minor amino acid differences of the deduced flavonone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) between both varieties were observed. The aurantiaca petals expressed substantially higher levels of transcripts representing CHS, F3H, DFR, ANS and UDP-glucose:flavonoid-3-O-glucosyltransferase genes, compared to lutea petals. Pelargonidin glycoside synthesis in aurantiaca petals therefore appears to reflect the higher steady-state levels of pelargonidin synthesis transcripts. Moreover, possible changes in the substrate specificity of DFR enzymes may represent additional mechanisms for producing red pelargonidin glycosides in petals of aurantiaca. Our report describing the exclusive accumulation of pelargonidin glycosides in aurantiaca petals may facilitate the modification of gentian flower color by the production of red anthocyanins. This work was supported by MICINN, Spain (BIO2014-54441-P, BIO2014-54426-P, BIO2013-44239-R, BIO2011-23324, BIO2011-22525, PIM2010PKB-00746); the National Natural Science Foundation of China (31270344); the Plan Nacional de I + D + I (2008-2011); the IBERCAROT network (112RT0445); PROGRAMA ESTATAL DE INVESTIGACIÓN CIENTÍFICA Y TÉCNICA DE EXCELENCIA, Spain (BIO2015-71703-REDT); COST action (EUROCAROTEN, OC-2015-1-19780); European Union Framework 7 European Research Council IDEAS Advanced Grant (to P.C.) ProgramBIOFORCE; and ERC Proof of Concept Grant (to P. C.), Catalan Government 2014 SGR 1296 Agricultural Biotechnology Research Group. T.V. was supported by a Ph.D. grant from the Plan Galego de Investigación e Crecemento 2011/2015 (Plan I2C), Consellería de Cultura, Educación e Ordenación Universitaria.

Multivitamin corn is a novel genetically engineered variety that simultaneously produces high levels of β-carotene, ascorbate and folate, and therefore has the potential to address simultaneously multiple micronutrient deficiencies caused by the lack of vitamins A, B9 and C in developing country populations. As part of the development process for genetically engineered crops and following European Food Safety Authority (EFSA) recommendations, multivitamin corn must be tested in whole food/feed sub-chronic animal feeding studies to ensure there are no adverse effects, and potential allergens must be identified. We carried out a 28-day toxicity assessment in mice, which showed no short-term sub-acute evidence of diet-related adverse health effects and no difference in clinical markers (food consumption, body weight, organ/tissue weight, haematological and biochemical blood parameters and histopathology) compared to mice fed on a control diet. A subsequent 90-day sub-chronic feeding study again showed no indications of toxicity compared to mice fed on control diets. Our data confirm that diets enriched with multivitamin corn have no adverse effects on mice, do not induce any clinical signs of toxicity and do not contain known allergens.

Genetic engineering (GE) is one of a raft of strategies that can be used to tackle malnutrition. Recent scientific advances have shown that multiple deficiencies can be tackled simultaneously using engineered plant varieties containing high levels of different minerals and organic nutrients. However, the impact of this progress is being diluted by the unwillingness of politicians to see beyond immediate popular support, favoring political expediency over controversial but potentially life-saving decisions based on rational scientific evidence. Research in our laboratory is supported by the Ministry of Science and Innovation, Spain (BFU2007-61413 and BIO2007-30738-E); European Research Council Advanced Grant (BIOFORCE) to PC; SmartCell, EU FP7 Integrated Project and COST ACTION FA0804. GF is supported through a PhD fellowship from the Spanish Ministry of Science and Innovation.

The eight Millennium Development Goals (MDGs) are international development targets for the year 2015 that aim to achieve relative improvements in the standards of health, socioeconomic status and education in the world’s poorest countries. Many of the challenges addressed by the MDGs reflect the direct or indirect consequences of subsistence agriculture in the developing world, and hence, plant biotechnology has an important role to play in helping to achieve MDG targets. In this opinion article, we discuss each of the MDGs in turn, provide examples to show how plant biotechnology may be able to accelerate progress towards the stated MDG objectives, and offer our opinion on the likelihood of such technology being implemented. In combination with other strategies, plant biotechnology can make a contribution towards sustainable development in the future although the extent to which progress can be made in today’s political climate depends on how we deal with current barriers to adoption. Research in our laboratory is supported by Ministry of Science and Innovation-MICINN, Spain (Grant BFU2007-61413); European Research Council Advanced Grant BIOFORCE; Center Consolider, MICINN, Spain; COST Action FA0804, Associated Unit CAVA and SmartCell, FP7 Integrated project.

Maize (also known as corn) is a domesticated cereal grain that has been grown as food and animal feed for tens of thousands of years. It is currently the most widely grown crop in the world, and is used not only for food/feed but also to produce ethanol, industrial starches and oils. Maize is now at the beginning of a new agricultural revolution, where the grains are used as factories to synthesize high-value molecules. In this article we look at the diversity of high-value products from maize, recent technological advances in the field and the emerging regulatory framework that governs how transgenic maize plants and their products are grown, used and traded. Research in our laboratory is supported by Ministry of Science and Innovation (MEI, Spain) Grant BFU2007-61413; the Ramon y Cajal program (MICINN, Spain); the Pharma-Planta FP6 EU project LSH-2002-1.2.5-2; Center Consolider, MICINN, Spain; Acciones Complementarias (MICINN, Spain) Grant BIO2005-24826; ERC Advanced Grant BIOFORCE to PC; SN and GF are recipients of MICINN PhD Fellowships.

Summary Lutein and zeaxanthin cannot be synthesized de novo in humans, and although lutein is abundant in fruit and vegetables, good dietary sources of zeaxanthin are scarce. Certain corn varieties provide adequate amounts because the ratio of endosperm β : e lycopene cyclase activity favours the β-carotene/zeaxanthin branch of the carotenoid pathway. We previously described a transgenic corn line expressing the early enzymes in the pathway (including lycopene β-cyclase) and therefore accumulating extraordinary levels of β-carotene. Here, we demonstrate that introgressing the transgenic mini-pathway into wild-type yellow endosperm varieties gives rise to hybrids in which the β : e ratio is altered additively. Where the β : e ratio in the genetic background is high, introgression of the mini-pathway allows zeaxanthin production at an unprecedented 56 μg/g dry weight. This result shows that metabolic synergy between endogenous and heterologous pathways can be used to enhance the levels of nutritionally important metabolites.