Generation and Evaluation of Modified \u3ci\u3eOpaque\u3c/i\u3e-2 Popcorn Suggests a Route to Quality Protein Popcorn

Introducing traits from dent corn to popcorn is challenging because it is difficult to recover adequate popping characteristics. QPM (Quality Protein Maize) is a dent corn variety carrying the opaque-2 (o2) mutation, specifying increased amounts of normally limiting essential amino acids, and modifier genes which restore the wild type vitreous kernel phenotype. In this study, we introgressed o2 and selected for endosperm modification using vitreousness and high 27-kD gamma zein content. In this way, we recovered high-lysine, fully poppable Quality Protein Popcorn (QPP). BC2F4 individuals with vitreous kernels were confirmed to be o2 mutants by both genotyping and SDSPAGE. Amino acid profiling of BC2F4 individuals showed that they all have significantly increased lysine compared with popcorn parental lines. Principal Component Analysis of the amino acid profiles showed that all introgressions were grouped with corresponding QPM parental lines. Popping analysis of the BC2F5 individuals showed that while there is variability in popping volume between lines, some lines show equivalent popping to the popcorn parent. In this proof-of-concept study for QPP, we have shown that it is possible to rapidly recover sufficient popcorn characteristics in a modified o2 background using simple phenotypic, biochemical and genetic selection. Furthermore, this shows increased y-zein is an acceptable substitute for a-zein for full poppability. Since we have developed multiple QPP introgressions, this gives good scope for ongoing hybrid production and future evaluation of agronomic performance and selection of elite hybrids. In a wider context, this study shows the potential for breeding beneficial traits into popcorn for agronomic improvement

Alteration of the interconversion of pyruvate and malate in the plastid or cytosol of ripening tomato fruit invokes diverse consequences on sugar but similar effects on cellular organic Acid, metabolism, and transitory starch accumulation

The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (NADP-ME) on tomato (Solanum lycopersicum) ripening. Transgenic tomato plants with strongly reduced levels of PEPCK and plastidic NADP-ME were generated by RNA interference gene silencing under the control of a ripening-specific E8 promoter. While these genetic modifications had relatively little effect on the total fruit yield and size, they had strong effects in fruit metabolism. Both transformants were characterized by lower levels of starch at breaker stage. Analysis of the activation state of ADP-glucose pyrophosphorylase correlated with the decrease of starch in both transformats, which suggest that is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positional labelled glucoses of fruits lacking in plastidic NADP-malic enzyme and cytosolic PEPCK activities revealed differential changes in overall respiration rates and tricarboxylic acid (TCA) cycle flux. Inactivation of cytosolic PEPCK affected the respiration rate which suggests that excess of oxaloacetate OAA is converted to aspartate and reintroduced in the TCA via 2-oxoglutarate/glutamate. On the other hand, the plastidic NADP-malic enzyme antisense lines were characterized by no changes in respiration rates and TCA cycle flux and together with an increase of pyruvate kinase and phosphoenolpyruvate carboxylase activities indicates that pyruvate is supply through these enzymes to the TCA cycle. These results are discussed in the context of current models of the importance of malate during tomato fruit ripening

Generation and Evaluation of Modified Opaque-2 Popcorn Suggests a Route to Quality Protein Popcorn

Introducing traits from dent corn to popcorn is challenging because it is difficult to recover adequate popping characteristics. QPM (Quality Protein Maize) is a dent corn variety carrying the opaque-2 (o2) mutation, specifying increased amounts of normally limiting essential amino acids, and modifier genes which restore the wild type vitreous kernel phenotype. In this study, we introgressed o2 and selected for endosperm modification using vitreousness and high 27-kD gamma zein content. In this way, we recovered high-lysine, fully poppable Quality Protein Popcorn (QPP). BC2F4 individuals with vitreous kernels were confirmed to be o2 mutants by both genotyping and SDS-PAGE. Amino acid profiling of BC2F4 individuals showed that they all have significantly increased lysine compared with popcorn parental lines. Principal Component Analysis of the amino acid profiles showed that all introgressions were grouped with corresponding QPM parental lines. Popping analysis of the BC2F5 individuals showed that while there is variability in popping volume between lines, some lines show equivalent popping to the popcorn parent. In this proof-of-concept study for QPP, we have shown that it is possible to rapidly recover sufficient popcorn characteristics in a modified o2 background using simple phenotypic, biochemical and genetic selection. Furthermore, this shows increased γ-zein is an acceptable substitute for α-zein for full poppability. Since we have developed multiple QPP introgressions, this gives good scope for ongoing hybrid production and future evaluation of agronomic performance and selection of elite hybrids. In a wider context, this study shows the potential for breeding beneficial traits into popcorn for agronomic improvement

Metabolic Profiling of a Mapping Population Exposes New Insights in the Regulation of Seed Metabolism and Seed, Fruit, and Plant Relations

To investigate the regulation of seed metabolism and to estimate the degree of metabolic natural variability, metabolite profiling and network analysis were applied to a collection of 76 different homozygous tomato introgression lines (ILs) grown in the field in two consecutive harvest seasons. Factorial ANOVA confirmed the presence of 30 metabolite quantitative trait loci (mQTL). Amino acid contents displayed a high degree of variability across the population, with similar patterns across the two seasons, while sugars exhibited significant seasonal fluctuations. Upon integration of data for tomato pericarp metabolite profiling, factorial ANOVA identified the main factor for metabolic polymorphism to be the genotypic background rather than the environment or the tissue. Analysis of the coefficient of variance indicated greater phenotypic plasticity in the ILs than in the M82 tomato cultivar. Broad-sense estimate of heritability suggested that the mode of inheritance of metabolite traits in the seed differed from that in the fruit. Correlation-based metabolic network analysis comparing metabolite data for the seed with that for the pericarp showed that the seed network displayed tighter interdependence of metabolic processes than the fruit. Amino acids in the seed metabolic network were shown to play a central hub-like role in the topology of the network, maintaining high interactions with other metabolite categories, i.e., sugars and organic acids. Network analysis identified six exceptionally highly co-regulated amino acids, Gly, Ser, Thr, Ile, Val, and Pro. The strong interdependence of this group was confirmed by the mQTL mapping. Taken together these results (i) reflect the extensive redundancy of the regulation underlying seed metabolism, (ii) demonstrate the tight co-ordination of seed metabolism with respect to fruit metabolism, and (iii) emphasize the centrality of the amino acid module in the seed metabolic network. Finally, the study highlights the added value of integrating metabolic network analysis with mQTL mapping

Techonolgy of Qualea grandiflora Mart. (Vochysiaceae) seeds

Qualea grandiflora Mart. (Vochysiaceae), commonly known as "pau-terra", is an arborous species native to the Brazilian savannah which possess commercial interests, as it can be used either as an ornamental or as a medicinal plant. "Pau-terra" can also be used in the heterogeneous reforestation of areas which are destined for restoration of permanent preservation degraded areas. Propagation studies with this species are scarce, being necessary then further clarification regarding the factors that influences the germination process. In this context, the objective of this work was to evaluate the influence of different temperatures, substrates and light conditions on seed germination. We selected light brown seeds which were subjected to different interactions between temperatures (15-25, 20-30, 25 and 30°C), substrate (paper, sand and vermiculite) and light (light and dark). All seeds were later dry-incubated at 32°C for 3, 6 and 12 hours. After treatments, seeds were kept in BOD at 58% RH and the following parameters were calculated: germination (%G) and germination speed index (GSI); the formation of normal and abnormal seedlings and the number dead seeds. Interaction was observed for all variables. In the optimum temperature range, the seeds behaved as photoblastic neutral or indifferent. Under alternating temperatures, darkness enhanced the germination, especially when combined with the lower temperatures. We noted that the sowing in sand, at 25°C, allowed the maintenance of suitable combinations of germination and seedling development. With respect to desiccation tolerance, "pau-terra" seeds presented an orthodox behavior, with a linear increase of the vigor as function of drying

The genetic architecture of branched-chain amino acid accumulation in tomato fruits

Previous studies of the genetic architecture of fruit metabolic composition have allowed us to identify four strongly conserved co-ordinate quantitative trait loci (QTL) for the branched-chain amino acids (BCAAs). This study has been extended here to encompass the other 23 enzymes described to be involved in the pathways of BCAA synthesis and degradation. On coarse mapping the chromosomal location of these enzymes, it was possible to define the map position of 24 genes. Of these genes eight co-localized, or mapped close to BCAA QTL including those encoding ketol-acid reductoisomerase (KARI), dihydroxy-acid dehydratase (DHAD), and isopropylmalate dehydratase (IPMD). Quantitative evaluation of the expression levels of these genes revealed that the S. pennellii allele of IPMD demonstrated changes in the expression level of this gene, whereas those of KARI and DHAD were invariant across the genotypes. Whilst the antisense inhibition of IPMD resulted in increased BCAA, the antisense inhibition of neither KARI nor DHAD produced a clear effect in fruit BCAA contents. The results are discussed both with respect to the roles of these specific enzymes within plant amino acid metabolism and within the context of current understanding of the regulation of plant branched-chain amino acid metabolism

The Re-Establishment of Desiccation Tolerance in Germinated Arabidopsis thaliana Seeds and Its Associated Transcriptome

The combination of robust physiological models with “omics” studies holds promise for the discovery of genes and pathways linked to how organisms deal with drying. Here we used a transcriptomics approach in combination with an in vivo physiological model of re-establishment of desiccation tolerance (DT) in Arabidopsis thaliana seeds. We show that the incubation of desiccation sensitive (DS) germinated Arabidopsis seeds in a polyethylene glycol (PEG) solution re-induces the mechanisms necessary for expression of DT. Based on a SNP-tile array gene expression profile, our data indicates that the re-establishment of DT, in this system, is related to a programmed reversion from a metabolic active to a quiescent state similar to prior to germination. Our findings show that transcripts of germinated seeds after the PEG-treatment are dominated by those encoding LEA, seed storage and dormancy related proteins. On the other hand, a massive repression of genes belonging to many other classes such as photosynthesis, cell wall modification and energy metabolism occurs in parallel. Furthermore, comparison with a similar system for Medicago truncatula reveals a significant overlap between the two transcriptomes. Such overlap may highlight core mechanisms and key regulators of the trait DT. Taking into account the availability of the many genetic and molecular resources for Arabidopsis, the described system may prove useful for unraveling DT in higher plants

Gene coexpression clusters and putative regulatory elements underlying seed storage reserve accumulation in Arabidopsis

Abstract Background In Arabidopsis, a large number of genes involved in the accumulation of seed storage reserves during seed development have been characterized, but the relationship of gene expression and regulation underlying this physiological process remains poorly understood. A more holistic view of this molecular interplay will help in the further study of the regulatory mechanisms controlling seed storage compound accumulation. Results We identified gene coexpression networks in the transcriptome of developing Arabidopsis (Arabidopsis thaliana) seeds from the globular to mature embryo stages by analyzing publicly accessible microarray datasets. Genes encoding the known enzymes in the fatty acid biosynthesis pathway were found in one coexpression subnetwork (or cluster), while genes encoding oleosins and seed storage proteins were identified in another subnetwork with a distinct expression profile. In the triacylglycerol assembly pathway, only the genes encoding diacylglycerol acyltransferase 1 (DGAT1) and a putative cytosolic "type 3" DGAT exhibited a similar expression pattern with genes encoding oleosins. We also detected a large number of putative cis-acting regulatory elements in the promoter regions of these genes, and promoter motifs for LEC1 (LEAFY COTYLEDON 1), DOF (DNA-binding-with-One-Finger), GATA, and MYB transcription factors (TF), as well as SORLIP5 (Sequences Over-Represented in Light-Induced Promoters 5), are overrepresented in the promoter regions of fatty acid biosynthetic genes. The conserved CCAAT motifs for B3-domain TFs and binding sites for bZIP (basic-leucine zipper) TFs are enriched in the promoters of genes encoding oleosins and seed storage proteins. Conclusions Genes involved in the accumulation of seed storage reserves are expressed in distinct patterns and regulated by different TFs. The gene coexpression clusters and putative regulatory elements presented here provide a useful resource for further experimental characterization of protein interactions and regulatory networks in this process.</p

Genome-wide association mapping identifies a new arsenate reductase enzyme critical for limiting arsenic accumulation in plants

Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the arsenate reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional arsenate reductase confirmed the arsenate reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to arsenate toxicity. We also confirmed the previous observation that the ACR2 arsenate reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1 single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing food such as rice

An integrated omics analysis reveals molecular mechanisms that are associated with differences in seed oil content between Glycine max and Brassica napus

Abstract Background: Rapeseed (Brassica napus L.) and soybean (Glycine max L.) seeds are rich in both protein and oil, which are major sources of biofuels and nutrition. Although the difference in seed oil content between soybean (~ 20%) and rapeseed (~ 40%) exists, little is known about its underlying molecular mechanism. Results: An integrated omics analysis was performed in soybean, rapeseed, Arabidopsis (Arabidopsis thaliana L. Heynh), and sesame (Sesamum indicum L.), based on Arabidopsis acyl-lipid metabolism- and carbon metabolism-related genes. As a result, candidate genes and their transcription factors and microRNAs, along with phylogenetic analysis and co-expression network analysis of the PEPC gene family, were found to be largely associated with the difference between the two species. First, three soybean genes (Glyma.13G148600, Glyma.13G207900 and Glyma.12G122900) co-expressed with GmPEPC1 are specifically enriched during seed storage protein accumulation stages, while the expression of BnPEPC1 is putatively inhibited by bna-miR169, and two genes BnSTKA and BnCKII are co-expressed with BnPEPC1 and are specifically associated with plant circadian rhythm, which are related to seed oil biosynthesis. Then, in de novo fatty acid synthesis there are rapeseed-specific genes encoding subunits β-CT (BnaC05g37990D) and BCCP1 (BnaA03g06000D) of heterogeneous ACCase, which could interfere with synthesis rate, and β-CT is positively regulated by four transcription factors (BnaA01g37250D, BnaA02g26190D, BnaC01g01040D and BnaC07g21470D). In triglyceride synthesis, GmLPAAT2 is putatively inhibited by three miRNAs (gma-miR171, gma-miR1516 and gma-miR5775). Finally, in rapeseed there was evidence for the expansion of gene families, CALO, OBO and STERO, related to lipid storage, and the contraction of gene families, LOX, LAH and HSI2, related to oil degradation. Conclusions: The molecular mechanisms associated with differences in seed oil content provide the basis for future breeding efforts to improve seed oil content