Essential amino acids in the Plant-Conserved and Class-Specific Regions of cellulose synthases.

The Plant-Conserved Region (P-CR) and the Class-Specific Region (CSR) are two plant-unique sequences in the catalytic core of cellulose synthases (CESAs) for which specific functions have not been established. Here, we used site-directed mutagenesis to replace amino acids and motifs within these sequences predicted to be essential for assembly and function of CESAs. We developed an in vivo method to determine the ability of mutated CesA1 transgenes to complement an Arabidopsis (Arabidopsis thaliana) temperature-sensitive root-swelling1 (rsw1) mutant. Replacement of a Cys residue in the CSR, which blocks dimerization in vitro, rendered the AtCesA1 transgene unable to complement the rsw1 mutation. Examination of the CSR sequences from 33 diverse angiosperm species showed domains of high-sequence conservation in a class-specific manner but with variation in the degrees of disorder, indicating a nonredundant role of the CSR structures in different CESA isoform classes. The Cys residue essential for dimerization was not always located in domains of intrinsic disorder. Expression of AtCesA1 transgene constructs, in which Pro417 and Arg453 were substituted for Ala or Lys in the coiled-coil of the P-CR, were also unable to complement the rsw1 mutation. Despite an expected role for Arg457 in trimerization of CESA proteins, AtCesA1 transgenes with Arg457Ala mutations were able to fully restore the wild-type phenotype in rsw1. Our data support that Cys662 within the CSR and Pro417 and Arg453 within the P-CR of Arabidopsis CESA1 are essential residues for functional synthase complex formation, but our data do not support a specific role for Arg457 in trimerization in native CESA complexes.

Natural variations of OsAUX5, a target gene of OsWRKY78, control the neutral essential amino acid content in rice grains.

Grain essential amino acid (EAA) levels contribute to rice nutritional quality. However, the molecular mechanisms underlying EAA accumulation and natural variation in rice grains remain unclear. Here we report the identification of a previously unrecognized auxin influx carrier subfamily gene, OsAUX5, which encodes an amino acid transporter that functions in uptake of multiple amino acids. We identified an elite haplotype of Pro::OsAUX5Hap2 that enhances grain EAA accumulation without an apparent negative effect on agronomic traits. Natural variations of OsAUX5 occur in the cis elements of its promoter, which are differentially activated because of the different binding affinity between OsWRKY78 and the W-box, contributing to grain EAA variation among rice varieties. The two distinct haplotypes were shown to have originated from different Oryza rufipogon progenitors, which contributed to the divergence between japonica and indica. Introduction of the indica-type Pro::OsAUX5Hap2 genotype into japonica could significantly increase EAA levels, indicating that indica-type Pro::OsAUX5Hap2 can be utilized to increase grain EAAs of japonica varieties. Collectively, our study uncovers an WRKY78-OsAUX5-based regulatory mechanism controlling grain EAA accumulation and provides a potential target for breeding EAA-rich rice.

Coevolution of Metabolic Pathways in Blattodea and Their Blattabacterium Endosymbionts, and Comparisons with Other Insect-Bacteria Symbioses.

Many insects harbor bacterial endosymbionts that supply essential nutrients and enable their hosts to thrive on a nutritionally unbalanced diet. Comparisons of the genomes of endosymbionts and their insect hosts have revealed multiple cases of mutually-dependent metabolic pathways that require enzymes encoded in 2 genomes. Complementation of metabolic reactions at the pathway level has been described for hosts feeding on unbalanced diets, such as plant sap. However, the level of collaboration between symbionts and hosts that feed on more variable diets is largely unknown. In this study, we investigated amino acid and vitamin/cofactor biosynthetic pathways in Blattodea, which comprises cockroaches and termites, and their obligate endosymbiont Blattabacterium cuenoti (hereafter Blattabacterium). In contrast to other obligate symbiotic systems, we found no clear evidence of "collaborative pathways" for amino acid biosynthesis in the genomes of these taxa, with the exception of collaborative arginine biosynthesis in 2 taxa, Cryptocercus punctulatus and Mastotermes darwiniensis. Nevertheless, we found that several gaps specific to Blattabacterium in the folate biosynthetic pathway are likely to be complemented by their host. Comparisons with other insects revealed that, with the exception of the arginine biosynthetic pathway, collaborative pathways for essential amino acids are only observed in phloem-sap feeders. These results suggest that the host diet is an important driving factor of metabolic pathway evolution in obligate symbiotic systems. IMPORTANCE The long-term coevolution between insects and their obligate endosymbionts is accompanied by increasing levels of genome integration, sometimes to the point that metabolic pathways require enzymes encoded in two genomes, which we refer to as "collaborative pathways". To date, collaborative pathways have only been reported from sap-feeding insects. Here, we examined metabolic interactions between cockroaches, a group of detritivorous insects, and their obligate endosymbiont, Blattabacterium, and only found evidence of collaborative pathways for arginine biosynthesis. The rarity of collaborative pathways in cockroaches and Blattabacterium contrasts with their prevalence in insect hosts feeding on phloem-sap. Our results suggest that host diet is a factor affecting metabolic integration in obligate symbiotic systems.

Genetic engineering to improve essential and conditionally essential amino acids in maize: transporter engineering as a reference.

Ruminants and humans are unable to synthesize essential amino acids (EAAs) and conditionally essential amino acids (CEAAs) under normal conditions and need to acquire them from plant sources. Maize plays, as a major crop, a central role in global food security. However, maize is deficient in several EAAs and CEAAs. Genetic engineering has been successfully used to enrich the EAA content of maize to some extent, including the content of Lys, Trp, and Met. However, research on other EAAs is lacking. Genetic engineering provides several viable approaches for increasing the EAA content in maize, including transformation of a single gene, transformation of multiple genes in a single cassette, overexpression of putative amino acid transporters, engineering the amino acid biosynthesis pathway including silencing of feedback inhibition enzymes, and overexpression of major enzymes in this pathway. These challenging processes require a deep understanding of the biosynthetic and metabolic pathways of individual amino acids, and the interaction of individual amino acids with other metabolic pathways.

Host and symbiont genetic determinants of nutritional phenotype in a natural population of the pea aphid.

A defining feature of the nutritional ecology of plant sap-feeding insects is that the dietary deficit of essential amino acids (EAAs) in plant sap is supplemented by EAA-provisioning microbial symbionts in the insect. Here, we demonstrated substantial variation in the nutritional phenotype of 208 genotypes of the pea aphid Acyrthosiphon pisum collected from a natural population. Specifically, the genotypes varied in performance (larval growth rates) on four test diets lacking the EAAs arginine, histidine and methionine or aromatic EAAs (phenylalanine and tryptophan), relative to the diet containing all EAAs. These data indicate that EAA supply from the symbiotic bacteria Buchnera can meet total aphid nutritional demand for only a subset of the EAA/aphid genotype combinations. We then correlated single nucleotide polymorphisms (SNPs) identified in the aphid and Buchnera genomes by reduced genome sequencing against aphid performance for each EAA deletion diet. This yielded significant associations between performance on the histidine-free diet and Buchnera SNPs, including metabolism genes predicted to influence histidine biosynthesis. Aphid genetic correlates of performance were obtained for all four deletion diets, with associations on the arginine-free diet and aromatic-free diets dominated by genes functioning in the regulation of metabolic and cellular processes. The specific aphid genes associated with performance on different EAA deletion diets are largely nonoverlapping, indicating some independence in the regulatory circuits determining aphid phenotype for the different EAAs. This study demonstrates how variation in the phenotype of associations collected from natural populations can be applied to elucidate the genetic basis of ecologically important traits in systems intractable to traditional forward/reverse genetic techniques.

Fortifying Horticultural Crops with Essential Amino Acids: A Review.

To feed the world's growing population, increasing the yield of crops is not the only important factor, improving crop quality is also important, and it presents a significant challenge. Among the important crops, horticultural crops (particularly fruits and vegetables) provide numerous health compounds, such as vitamins, antioxidants, and amino acids. Essential amino acids are those that cannot be produced by the organism and, therefore, must be obtained from diet, particularly from meat, eggs, and milk, as well as a variety of plants. Extensive efforts have been devoted to increasing the levels of essential amino acids in plants. Yet, these efforts have been met with very little success due to the limited genetic resources for plant breeding and because high essential amino acid content is generally accompanied by limited plant growth. With a deep understanding of the biosynthetic pathways of essential amino acids and their interactions with the regulatory networks in plants, it should be possible to use genetic engineering to improve the essential amino acid content of horticultural plants, rendering these plants more nutritionally favorable crops. In the present report, we describe the recent advances in the enhancement of essential amino acids in horticultural plants and possible future directions towards their bio-fortification.

Broadening Our Portfolio in the Genetic Improvement of Maize Chemical Composition.

The adoption of recombinant inbred line and introgression line populations, as well as the study of association mapping panels, has greatly accelerated our ability to identify the genes underlying plant phenotypic variance. In tandem, the development of metabolomics approaches has greatly enhanced our ability to comprehensively define cellular chemical composition. As a consequence, breeding for chemical composition is being extended beyond our traditional targets of oil and protein to include components such as essential amino acids, vitamins, and antioxidant secondary metabolites with considerable purported consequences for human health. Here, we review the above-mentioned developments paying particular attention to the genetic architecture of metabolic traits as well as updating the perspective for utilizing metabolomics in maize improvement.

Expression of threonine-biosynthetic genes in mammalian cells and transgenic mice.

Threonine is a nutritionally essential amino acid (EAA) for the growth and development of humans and other nonruminant animals and must be provided in diets to sustain life. The aim of this study was to synthesize threonine in mammalian cells through transgenic techniques. To achieve this goal, we combined the genes involved in bacterial threonine biosynthesis pathways into a single open reading frame separated by self-cleaving peptides (2A) and then linked it into a transposon system (piggyBac). The plasmids pEF1a-IRES-GFP-E2F-his and pEF1a-IRES-GFP-M2F-his expressed Escherichia coli homoserine kinase and threonine synthase efficiently in mouse cells and enabled cells to synthesize threonine from homoserine. This biosynthetic pathway occurred with a low level of efficiency in transgenic mice. Three transgenic mice were identified by Southern blot from 72 newborn mice, raising the possibility that a high level of expression of these genes in mouse embryos might be lethal. The results indicated that it is feasible to synthesize threonine in animal cells using genetic engineering technology. Further work is required to improve the efficiency of this method for introducing genes into mammals. We propose that the transgenic technology provides a promising means to enhance the synthesis of nutritionally EAAs in farm animals and to eliminate or reduce supplementation of these nutrients in diets for livestock, poultry and fish.

QTL mapping based on different genetic systems for essential amino acid contents in cottonseeds in different environments.

Cottonseeds are rich in various essential amino acids. However, the inheritance of them at molecular level are still not defined across various genetic systems. In the present study, using a newly developed mapping model that can analyze the embryo and maternal main effects as well as QTL × environment interaction effects on quantitative quality trait loci (QTLs) in cottonseeds, a study on QTL located in the tetraploid embryo and tetraploid maternal plant genomes for essential amino acid contents in cottonseeds under different environments was carried out, using the immortal F2 (IF2) populations from a set of 188 recombinant inbred lines derived from an intraspecific hybrid cross of two upland cotton germplasms HS46 and MARKCBUCAG8US-1-88 as experimental materials. The results showed a total of 35 QTLs associated with these quality traits in cottonseeds. Nineteen QTLs were subsequently mapped on chromosome 5, 6 and 8 in sub-A genome and chromosome 15, 18, 22 and 23 in sub-D genome. Eighteen QTLs were also found having QTL × environment (QE) interaction effects. The genetic main effects from QTLs located on chromosomes in the embryo and maternal plant genomes and their QE effects in different environments were all important for these essential amino acids in cottonseeds. The results suggested that the influence of environmental factors on the expression of some QTLs located in different genetic systems should be considered when improving for these amino acids. This study can serve as the foundation for the improvement of these essential amino acids in cottonseeds.

Nutritional enhancement of rice for human health: the contribution of biotechnology.

Micronutrient malnutrition is widespread, especially in poor populations across the globe where daily caloric intake is confined mainly to staple cereals. Rice, which is a staple food for over half of the world's population, is low in bioavailable micronutrients required for the daily diet. Improvements of the plant-based diets are therefore critical and of high economic value in order to achieve a healthy nutrition of a large segment of the human population. Rice grain biofortification has emerged as a strategic priority for alleviation of micronutrient malnutrition. Nutritional enhancement of crops through conventional breeding is often limited by the low genetic variability for required dietary micronutrient levels. In this case, biotechnology strategies offer effective and efficient perspectives. In this review, we discuss genetic engineering approaches that have been successful in the nutritional enhancement of rice endosperm. These advancements will make substantial contributions to crop improvement and human nutrition. Their practical application, however, also demands visionary changes in regulatory policies and a broader consumer acceptance.

Cellular and animal indispensable amino acid limitation responses and health promotion. Can the two be linked? A critical review.

Cellular growth repression can mediate positive health outcomes by improving resistance while delaying the manifestation and decelerating the progression, of chronic diseases. Sensing systems that respond to amino acid limitation are, the general control non-derepressible kinase 2 (GCN2), the mammalian target of rapamycin (mTOR; namely mammalian target of rapamycin complex 1), the extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase pathway, the adenosine 5-mono-phosphate-activated protein kinase system. GCN2 particularly, under limiting essential amino acid conditions, activates the integrated stress response (ISR) causing selective up- /down-regulation of pro-survival/pro-apoptotic genes, respectively, rendering beneficial adaptation responses to amino acid limitation. This review attempts to bridge the link between molecular events and mechanisms observed at the cellular level with the potential health benefits possibly achieved at the whole organism level. The article describes mechanisms of essential amino acid sensing and provides a discussion on relevant research that suggests a potential role of essential amino acid sensing for promoting health.

Newly identified essential amino acid residues affecting Δ8-sphingolipid desaturase activity revealed by site-directed mutagenesis.

In order to identify amino acid residues crucial for the enzymatic activity of Δ(8)-sphingolipid desaturases, a sequence comparison was performed among Δ(8)-sphingolipid desaturases and Δ(6)-fatty acid desaturases from various plants. In addition to the known conserved cytb(5) (cytochrome b(5)) HPGG motif and three conserved histidine boxes, they share additional 15 completely conserved residues. A series of site-directed mutants were generated using our previously isolated Δ(8)-sphingolipid desaturase gene from Brassica rapa to evaluate the importance of these residues to the enzyme function. The mutants were functionally characterized by heterologous expression in yeast, allowing the identification of the products of the enzymes. The results revealed that residues H63, N203, D208, D210, and G368 were obligatorily required for the enzymatic activity, and substitution of the residues F59, W190, W345, L369 and Q372 markedly decreased the enzyme activity. Among them, replacement of the residues W190, L369 and Q372 also has significant influence on the ratio of the two enzyme products. Information obtained in this work provides the molecular basis for the Δ(8)-sphingolipid desaturase activity and aids in our understanding of the structure-function relationships of the membrane-bound desaturases.

Multimodal dynamic response of the Buchnera aphidicola pLeu plasmid to variations in leucine demand of its host, the pea aphid Acyrthosiphon pisum.

Aphids, important agricultural pests, can grow and reproduce thanks to their intimate symbiosis with the γ-proteobacterium Buchnera aphidicola that furnishes them with essential amino acids lacking in their phloem sap diet. To study how B. aphidicola, with its reduced genome containing very few transcriptional regulators, responds to variations in the metabolic requirements of its host, we concentrated on the leucine metabolic pathway. We show that leucine is a limiting factor for aphid growth and it displays a stimulatory feeding effect. Our metabolic analyses demonstrate that symbiotic aphids are able to respond to leucine starvation or excess by modulating the neosynthesis of this amino acid. At a molecular level, this response involves an early important transcriptional regulation (after 12 h of treatment) followed by a moderate change in the pLeu plasmid copy number. Both responses are no longer apparent after 7 days of treatment. These experimental data are discussed in the light of a re-annotation of the pLeu plasmid regulatory elements. Taken together, our data show that the response of B. aphidicola to the leucine demand of its host is multimodal and dynamically regulated, providing new insights concerning the genetic regulation capabilities of this bacterium in relation to its symbiotic functions.

Analysis of embryo, cytoplasmic and maternal genetic correlations for seven essential amino acids in rapeseed meal (Brassica napus L.).

Genetic correlations of nutrient quality traits including lysine, methionine, leucine, isoleucine, phenylalanine, valine and threonine contents in rapeseed meal were analysed by the genetic model for quantitative traits of diploid plants using a diallel design with nine parents of Brassica napus L. These results indicated that the genetic correlations of embryo, cytoplasm and/or maternal plant havemade different contribution to total genetic correlations of most pairwise nutrient quality traits. The genetic correlations among the amino acids in rapeseed meal were simultaneously controlled by genetic main correlations and genotype x environment (GE) interaction correlations, especially for the maternal dominance correlations. Most components of genetic main correlations and GE interaction correlations for the pairwise traits studied were significantly positive. Some of the pairwise traits had negative genetic correlations, especially between valine and other amino acid contents. Indirect selection for improving the quality traits of rapeseed meal could be expected in rape breeding according to the magnitude and direction of genetic correlation components.

Aphid genome expression reveals host-symbiont cooperation in the production of amino acids.

The evolution of intimate symbiosis requires the coordination of gene expression and content between the distinct partner genomes; this coordination allows the fusion of capabilities of each organism into a single integrated metabolism. In aphids, the 10 essential amino acids are scarce in the phloem sap diet and are supplied by the obligate bacterial endosymbiont (Buchnera), which lives inside specialized cells called bacteriocytes. Although Buchnera's genome encodes most genes for essential amino acid biosynthesis, several genes in essential amino acid pathways are missing, as are most genes for production of nonessential amino acids. Additionally, it is unresolved whether the supply of nitrogen for amino acid biosynthesis is supplemented by recycling of waste ammonia. We compared pea aphid gene expression between bacteriocytes and other body tissues using RNA sequencing and pathway analysis and exploiting the genome sequences available for both partners. We found that 26 genes underlying amino acid biosynthesis were up-regulated in bacteriocytes. Seven of these up-regulated genes fill the gaps of Buchnera's essential amino acid pathways. In addition, genes underlying five nonessential amino acid pathways lost from Buchnera are up-regulated in bacteriocytes. Finally, our results reveal that two genes, glutamine synthetase and glutamate synthase, which potentially work together in the incorporation of ammonium nitrogen into glutamate (GOGAT) cycle to assimilate ammonia into glutamate, are up-regulated in bacteriocytes. Thus, host gene expression and symbiont capabilities are closely integrated within bacteriocytes, which function as specialized organs of amino acid production. Furthermore, the GOGAT cycle may be a key source of nitrogen fueling the integrated amino acid metabolism of the aphid-Buchnera partnership.

Analysis of the Yersinia enterocolitica PspBC proteins defines functional domains, essential amino acids and new roles within the phage-shock-protein response.

The Yersinia enterocolitica phage-shock-protein (Psp) stress response system is activated by mislocalized outer-membrane secretin components of protein export systems and is essential for virulence. The cytoplasmic membrane proteins PspB and PspC were proposed to be dual function components of the system, acting both as positive regulators of psp gene expression and to support survival during secretin-induced stress. In this study we have uncoupled the regulatory and physiological functions of PspBC and discovered unexpected new roles, functional domains and essential amino acids. First, we showed that PspB controls PspC concentration by both pre- and post-transcriptional mechanisms. We then screened for PspBC mutants with altered transcriptional regulatory function. Unexpectedly, we identified PspB and PspC mutants that activated psp gene expression in the absence of secretin-induced stress. Together with a subsequent truncation analysis, this revealed that the PspC cytoplasmic domain plays an unforeseen role in negatively regulating psp gene expression. Conversely, mutations within the PspC periplasmic domain abolished its ability to activate psp gene expression. Significantly, PspC mutants unable to activate psp gene expression retained their ability to support survival during secretin-induced stress. These data provide compelling support for the proposal that these two functions are independent.

Enhancing essential amino acids and health benefit components in grain crops for improved nutritional values.

Improving essential amino acids or protein content, along with other phytonutrients in the food crops, will affect a great portion of the world population, especially in developing countries where rice grain is the main source of protein. Malnutrition, including deficiencies in protein/energy, iron/zinc, vitamin A, and iodine, causes a total 24,000 deaths per day worldwide. The problem is severe where rice is the major staple food. Protein deficiency involves both the quantity (amount) and quality (the content in essential amino acids) of the dietary protein. Various interventions, such as distribution, fortification, dietary diversification, and measures against infectious diseases, have been applied to reduce deficiency disorders. The problem, however, remains unsolved. Developing genetically novel lines with elevated content of essential amino acids together with other health benefit components becomes more feasible for the enhancement of breeding techniques, genomics, molecular manipulations, and genetic engineering. Advancement in basic genetic and genetic engineering has resulted in successful enrichment of some essential amino acids, such as lysine (Lys), tryptophan (Trp), and methionine (Met). Successful genetic enhancement has been largely restricted to the maize crop through enrichment of grain Lys and to some extends Trp. Since rice is the main source of calories and protein intake for billions of people, enhancing essential amino acids in rice represents a tremendous challenge. This paper will discuss and review the current status in basic genetics, molecular genetics, and genetic engineering associated with the enhancement of amino acids and other health benefit components in major grain crop improvement. Patents and future efforts associated with enhancing nutritional quality of the grain will also be reviewed as a concerted effort to solve the malnutrition problem and improve the quality of life worldwide.

Transgenic approach to improve wheat (Triticum aestivum L.) nutritional quality.

An amaranth (Amaranthus hypochondriacus) albumin gene, encoding the 35-kDa AmA1 protein of the seed, with a high content of essential amino acids, was used in the biolistic transformation of bread wheat (Triticum aestivum L.) variety Cadenza. The transformation cassette carried the ama1 gene under the control of a powerful wheat endosperm-specific promoter (1Bx17 HMW-GS). Southern-blot analysis of T(1) lines confirmed the integration of the foreign gene, while RT-PCR and Western-blot analyses of the samples confirmed the transcription and translation of the transgene. The effects of the extra albumin protein on the properties of flour, produced from bulked T(2) seeds, were calculated using total protein and essential amino acid content analysis, polymeric/monomeric protein and HMW/LMW glutenin subunit ratio measurements. The results indicated that not only can essential amino acid content be increased, but some parameters associated with functional quality may also be improved because of the expression of the AmA1 protein.

Essential amino acid usage and evolutionary nutrigenomics of eukaryotes--insights into the differential usage of amino acids in protein domains and extra-domains.

Nutrigenomics studies the effects of nutrients on the genome, transcriptome and proteome of organisms, and here an evolutionary standpoint on this new discipline is presented. It is well known that metazoan organisms are unable to synthesize all amino acids necessary to produce their proteins and that these essential amino acids (EAA) must be acquired from the diet. Here, we tested the hypothesis that conserved regions such as protein domains (DM) have different essentiality indexes and use different sets of amino acids when compared to extra-domains (ED) and proteins without mapped domains (WD). We found that auxotrophic organisms have a tendency to use less EAAs in DM than do prototrophic ones. Looking into the amino acid usage of eukaryotic proteins downloaded from KEGG and COG, we showed that WD have a usage of amino acids closer to DM, which suggests that proteins without mapped domains behave as large domains. Using an ED index that shows the proportion of prevalent amino acids in ED, a differential usage of amino acids in domains versus extra-domains was demonstrated. Protein domains were shown to be enriched with a higher number of EAA, and it may be related to the fact that these amino acids had lost their biosynthetic pathways in metazoans during a great amino acid pathway deletion, followed by a nutritional constraint that may have happened close to the conquest of the terrestrial environment. Thus, the proportion of EAA outside domains could have decreased during evolution due to nutritional constraints.