Publications by authors named "Ruthie Angelovici"

32 Publications

Genetic variation, environment and demography intersect to shape Arabidopsis defense metabolite variation across Europe.

Elife 2021 May 5;10. Epub 2021 May 5.

Department of Plant Sciences, University of California, Davis, Davis, United States.

Plants produce diverse metabolites to cope with the challenges presented by complex and ever-changing environments. These challenges drive the diversification of specialized metabolites within and between plant species. However, we are just beginning to understand how frequently new alleles arise controlling specialized metabolite diversity and how the geographic distribution of these alleles may be structured by ecological and demographic pressures. Here, we measure the variation in specialized metabolites across a population of 797 natural accessions. We show that a combination of geography, environmental parameters, demography and different genetic processes all combine to influence the specific chemotypes and their distribution. This showed that causal loci in specialized metabolism contain frequent independently generated alleles with patterns suggesting potential within-species convergence. This provides a new perspective about the complexity of the selective forces and mechanisms that shape the generation and distribution of allelic variation that may influence local adaptation.
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http://dx.doi.org/10.7554/eLife.67784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8205490PMC
May 2021

Final Selection of Quality Protein Popcorn Hybrids.

Front Plant Sci 2021 24;12:658456. Epub 2021 Mar 24.

Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE, United States.

Quality Protein Popcorn (QPP) BCF inbred lines were produced through an interpopulation breeding system between Quality Protein Maize dent (QPM) and elite popcorn germplasm. In 2019, five QPP F hybrids were selected for further evaluation due to superior agronomics, endosperm protein quality, and popping quality traits. Though these BCF QPP hybrids were phenotypically similar to their popcorn parents, the QPP cultivars conveyed slightly inferior popping characteristics when compared to the original popcorn germplasm. The objective of this study was twofold. First, BCF inbred lines were crossed to their popcorn parents and BCF inbred lines were produced for hybridization to test the agronomic, protein, and popping trait effects from an additional QPP by popcorn backcross. Second, BC- and BC-hybrids were simultaneously evaluated alongside ConAgra Brands elite cultivars and ranked for potential commercialization in the spring of 2020. These 10 QPP hybrids were grown alongside five ConAgra Brands elite popcorn cultivars in three locations and agronomic, protein quality, and popping quality traits were evaluated. Significant improvements in popcorn quality traits were observed in the QPP BC cultivars compared to their BC counterparts, and yield averages were significantly lower in BC-derived QPP hybrids compared to the BC population. Protein quality traits were not significantly different between QPP backcrossing populations and significantly superior to ConAgra elite popcorn varieties. Utilizing a previously published ranking system, six QPP hybrids, three from the BCF population and three from the BCF population, were evaluated as candidates for final selection. The successful evaluation and ranking system methodology employed is transferable to other hybrid production and testing programs. Incorporating this analysis with concurrent sensory studies, two QPP hybrids were chosen as premier cultivars for potential commercialization.
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http://dx.doi.org/10.3389/fpls.2021.658456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025670PMC
March 2021

Genomic Prediction Informed by Biological Processes Expands Our Understanding of the Genetic Architecture Underlying Free Amino Acid Traits in Dry Seeds.

G3 (Bethesda) 2020 11 5;10(11):4227-4239. Epub 2020 Nov 5.

Division of Biological Sciences, University of Missouri, Columbia, MO

Plant growth, development, and nutritional quality depends upon amino acid homeostasis, especially in seeds. However, our understanding of the underlying genetics influencing amino acid content and composition remains limited, with only a few candidate genes and quantitative trait loci identified to date. Improved knowledge of the genetics and biological processes that determine amino acid levels will enable researchers to use this information for plant breeding and biological discovery. Toward this goal, we used genomic prediction to identify biological processes that are associated with, and therefore potentially influence, free amino acid (FAA) composition in seeds of the model plant Markers were split into categories based on metabolic pathway annotations and fit using a genomic partitioning model to evaluate the influence of each pathway on heritability explained, model fit, and predictive ability. Selected pathways included processes known to influence FAA composition, albeit to an unknown degree, and spanned four categories: amino acid, core, specialized, and protein metabolism. Using this approach, we identified associations for pathways containing known variants for FAA traits, in addition to finding new trait-pathway associations. Markers related to amino acid metabolism, which are directly involved in FAA regulation, improved predictive ability for branched chain amino acids and histidine. The use of genomic partitioning also revealed patterns across biochemical families, in which serine-derived FAAs were associated with protein related annotations and aromatic FAAs were associated with specialized metabolic pathways. Taken together, these findings provide evidence that genomic partitioning is a viable strategy to uncover the relative contributions of biological processes to FAA traits in seeds, offering a promising framework to guide hypothesis testing and narrow the search space for candidate genes.
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http://dx.doi.org/10.1534/g3.120.401240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642941PMC
November 2020

Deletion of maize RDM4 suggests a role in endosperm maturation as well as vegetative and stress-responsive growth.

J Exp Bot 2020 10;71(19):5880-5895

Department of Agronomy and Horticulture, Center for Plant Science Innovation, Beadle Center for Biotechnology, University of Nebraska, Lincoln, NE, USA.

Opaque kernels in maize may result from mutations in many genes, such as OPAQUE-2. In this study, a maize null mutant of RNA-DIRECTED DNA METHYLATION 4 (RDM4) showed an opaque kernel phenotype, as well as plant developmental delay, male sterility, and altered response to cold stress. We found that in opaque kernels, all zein proteins were reduced and amino acid content was changed, including increased lysine. Transcriptomic and proteomic analysis confirmed the zein reduction and proteomic rebalancing of non-zein proteins, which was quantitatively and qualitatively different from opaque-2. Global transcriptional changes were found in endosperm and leaf, including many transcription factors and tissue-specific expressed genes. Furthermore, of the more than 8000 significantly differentially expressed genes in wild type in response to cold, a significant proportion (25.9% in moderate cold stress and 40.8% in near freezing stress) were not differentially expressed in response to cold in rdm4, suggesting RDM4 may participate in regulation of abiotic stress tolerance. This initial characterization of maize RDM4 provides a basis for further investigating its function in endosperm and leaf, and as a regulator of normal and stress-responsive development.
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http://dx.doi.org/10.1093/jxb/eraa325DOI Listing
October 2020

Production and Selection of Quality Protein Popcorn Hybrids Using a Novel Ranking System and Combining Ability Estimates.

Front Plant Sci 2020 23;11:698. Epub 2020 Jun 23.

Department of Agronomy and Horticulture, University of Nebraska - Lincoln, Lincoln, NE, United States.

Popcorn varieties are agronomically sub-optimal and genetically limited compared to other maize subspecies. To increase genetic diversity and improve popcorn agronomics, dent germplasm has been introduced to popcorn with limited success and generally, major loss of popping. Between 2013 and 2018, 12 Quality Protein Popcorn (QPP) inbreds containing Quality Protein Maize (QPM) and popcorn germplasm were produced that maintained popping while carrying the allele conferring elevated kernel lysine. This is an opportune trait in the growing market for healthier snacks and a model for mining QPM traits into popcorn. We crossed QPP inbreds to explore the effects of heterosis on popcorn protein, popping quality, and plant agronomics and selected hybrids for further production. To rank and intermediately prescreen hybrids, we utilized a novel hybrid-ranking model adapted from a rank summation index while examining the inbred general combining ability and hybrid specific combining ability estimates for all traits. We observed a biological manifestation of heterosis by categorizing hybrids by pedigree that resulted in a stepwise progression of trait improvement. These results corroborated our hybrid selection and offered insight in basic heterosis research. Estimates for popcorn quality and agronomic trait covariances also suggest the synergistic introgression of highly vitreous dent maize (QPM) into popcorn, providing a likely explanation for the successfully maintained vitreous endosperm, protein quality and popping traits in line with a remodeled proteome. QPP hybrids maintained improved amino acid profiles although different popping methods variably affected popcorn's protein bound and free amino acid levels. This preliminary screening of QPP hybrids is enabling further quantitative selection for large-scale, complex trait comparison to currently marketed elite popcorn varieties.
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http://dx.doi.org/10.3389/fpls.2020.00698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325744PMC
June 2020

HAPPI GWAS: Holistic Analysis with Pre- and Post-Integration GWAS.

Bioinformatics 2020 11;36(17):4655-4657

Division of Biological Sciences, MU Institute for Data Science and Informatics, University of Missouri, Columbia, MO 65211, USA.

Motivation: Advanced publicly available sequencing data from large populations have enabled informative genome-wide association studies (GWAS) that associate SNPs with phenotypic traits of interest. Many publicly available tools able to perform GWAS have been developed in response to increased demand. However, these tools lack a comprehensive pipeline that includes both pre-GWAS analysis, such as outlier removal, data transformation and calculation of Best Linear Unbiased Predictions or Best Linear Unbiased Estimates. In addition, post-GWAS analysis, such as haploblock analysis and candidate gene identification, is lacking.

Results: Here, we present Holistic Analysis with Pre- and Post-Integration (HAPPI) GWAS, an open-source GWAS tool able to perform pre-GWAS, GWAS and post-GWAS analysis in an automated pipeline using the command-line interface.

Availability And Implementation: HAPPI GWAS is written in R for any Unix-like operating systems and is available on GitHub (https://github.com/Angelovici-Lab/HAPPI.GWAS.git).

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa589DOI Listing
November 2020

mGWAS Uncovers Gln-Glucosinolate Seed-Specific Interaction and its Role in Metabolic Homeostasis.

Plant Physiol 2020 06 21;183(2):483-500. Epub 2020 Apr 21.

Division of Biological Sciences, Interdisciplinary Plant Group, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65211

Gln is a key player in plant metabolism. It is one of the major free amino acids that is transported into the developing seed and is central for nitrogen metabolism. However, Gln natural variation and its regulation and interaction with other metabolic processes in seeds remain poorly understood. To investigate the latter, we performed a metabolic genome-wide association study (mGWAS) of Gln-related traits measured from the dry seeds of the Arabidopsis () diversity panel using all potential ratios between Gln and the other members of the Glu family as traits. This semicombinatorial approach yielded multiple candidate genes that, upon further analysis, revealed an unexpected association between the aliphatic glucosinolates (GLS) and the Gln-related traits. This finding was confirmed by an independent quantitative trait loci mapping and statistical analysis of the relationships between the Gln-related traits and the presence of specific GLS in seeds. Moreover, an analysis of Arabidopsis mutants lacking GLS showed an extensive seed-specific impact on Gln levels and composition that manifested early in seed development. The elimination of GLS in seeds was associated with a large effect on seed nitrogen and sulfur homeostasis, which conceivably led to the Gln response. This finding indicates that both Gln and GLS play key roles in shaping the seed metabolic homeostasis. It also implies that select secondary metabolites might have key functions in primary seed metabolism. Finally, our study shows that an mGWAS performed on dry seeds can uncover key metabolic interactions that occur early in seed development.
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http://dx.doi.org/10.1104/pp.20.00039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271782PMC
June 2020

Can metabolic tightening and expansion of co-expression network play a role in stress response and tolerance?

Plant Sci 2020 Apr 10;293:110409. Epub 2020 Jan 10.

Division of Biological Sciences, Interdisciplinary Plant Group, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, 65201, USA. Electronic address:

Plants respond and adapt to changes in their environment by employing a wide variety of genetic, molecular, and biochemical mechanisms. When so doing, they trigger large-scale rearrangements at the metabolic and transcriptional levels. The dynamics and patterns of these rearrangements and how they govern a stress response is not clear. In this opinion, we discuss a plant's response to stress from the perspective of the metabolic gene co-expression network and its rearrangement upon stress. As a case study, we use publicly available expression data of Arabidopsis thaliana plants exposed to heat and drought stress to evaluate and compare the co-expression networks of metabolic genes. The analysis highlights that stress conditions can lead to metabolic tightening and expansion of the co-expression network. We argue that this rearrangement could play a role in a plant's response to stress and thus may be an additional tool to assess and understand stress tolerance/sensitivity. Additional studies are needed to evaluate the metabolic network in response to multiple stresses at various intensities and across different genetic backgrounds (e.g., intra- and inter-species, sensitive and tolerant eco/genotypes).
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http://dx.doi.org/10.1016/j.plantsci.2020.110409DOI Listing
April 2020

The complex response of free and bound amino acids to water stress during the seed setting stage in Arabidopsis.

Plant J 2020 05 5;102(4):838-855. Epub 2020 Feb 5.

Division of Biological Sciences, Interdisciplinary Plant Group, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, 65211, USA.

Free amino acids (FAAs) and protein-bound amino acids (PBAAs) in seeds play an important role in seed desiccation, longevity, and germination. However, the effect that water stress has on these two functional pools, especially when imposed during the crucial seed setting stage is unclear. To better understand these effects, we exposed Arabidopsis plants at the seed setting stage to a range of water limitation and water deprivation conditions and then evaluated physiological, metabolic, and proteomic parameters, with special focus on FAAs and PBAAs. We found that in response to severe water limitation, seed yield decreased, while seed weight, FAA, and PBAA content per seed increased. Nevertheless, the composition of FAAs and PBAAs remained unaltered. In response to severe water deprivation, however, both seed yield and weight were reduced. In addition, major alterations were observed in both FAA and proteome compositions, which indicated that both osmotic adjustment and proteomic reprogramming occurred in these naturally desiccation-tolerant organs. However, despite the major proteomic alteration, the PBAA composition did not change, suggesting that the proteomic reprogramming was followed by a proteomic rebalancing. Proteomic rebalancing has not been observed previously in response to stress, but its occurrence under stress strongly suggests its natural function. Together, our data show that the dry seed PBAA composition plays a key role in seed fitness and therefore is rigorously maintained even under severe water stress, while the FAA composition is more plastic and adaptable to changing environments, and that both functional pools are distinctly regulated.
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http://dx.doi.org/10.1111/tpj.14668DOI Listing
May 2020

Adaptive responses of amino acid metabolism to the combination of desiccation and low nitrogen availability in Sporobolus stapfianus.

Planta 2019 May 6;249(5):1535-1549. Epub 2019 Feb 6.

Division of Biological Sciences, Interdisciplinary Plant Group, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, 65311, USA.

Main Conclusion: Depending on nitrogen availability, S. stapfianus uses different amino acid metabolism strategies to cope with desiccation stress. The different metabolic strategies support essential processes for the desiccation tolerance phenotype. To provide a comprehensive assessment of the role played by amino acids in the adaptation of Sporobolus stapfianus to a combination of desiccation and nitrogen limitation, we used an absolute quantification of free and protein-bound amino acids (FAAs and PBAAs) as well as their gamma-glutamyl (gg-AA) derivatives in four different tissues grown under high- and low-nitrogen regimes. We demonstrate that although specific FAAs and gg-AAs increased in desiccating immature leaves under both nitrogen regimes, the absolute change in the total amount of either is small or negligible, negating their proposed role in nitrogen storage. FAAs and PBAAs decrease in underground tissues during desiccation, when nitrogen is abundant. In contrast, PBAAs are drastically reduced from the mature leaves, when nitrogen is limiting. Nevertheless, the substantial reduction in PBAA and FAA fractions in both treatments is not manifested in the immature leaves, which strongly suggests that these amino acids are further metabolized to fuel central metabolism or other metabolic adjustments that are essential for the acquisition of desiccation tolerance (DT).
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http://dx.doi.org/10.1007/s00425-019-03105-6DOI Listing
May 2019

The renaissance of comparative biochemistry.

Am J Bot 2019 01 10;106(1):3-13. Epub 2019 Jan 10.

Department of Biochemistry and Center for Plant Biology, Purdue University, West Lafayette, IN, USA.

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http://dx.doi.org/10.1002/ajb2.1216DOI Listing
January 2019

Generation and Evaluation of Modified Popcorn Suggests a Route to Quality Protein Popcorn.

Front Plant Sci 2018 6;9:1803. Epub 2018 Dec 6.

Department of Agronomy and Horticulture, University of Nebraska - Lincoln, Lincoln, NE, United States.

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 () 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 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). BCF individuals with vitreous kernels were confirmed to be mutants by both genotyping and SDS-PAGE. Amino acid profiling of BCF 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 BCF 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 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.
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http://dx.doi.org/10.3389/fpls.2018.01803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6291453PMC
December 2018

A High-Throughput Absolute-Level Quantification of Protein-Bound Amino Acids in Seeds.

Curr Protoc Plant Biol 2018 12 8;3(4):e20084. Epub 2018 Nov 8.

Bond Life Sciences Center, Division of Biological Sciences, Interdisciplinary Plant Group, University of Missouri, Columbia, Missouri.

In this unit, we describe a high-throughput absolute quantification protocol for 16 protein-bound amino acids (PBAAs) that combines a microscale protein hydrolysis step and an absolute quantification step using multiple reaction monitoring-based liquid chromatography-tandem mass spectrometry detection. The approach facilitates analysis of a few hundred samples per week by using a 96-well-plate extraction setup and avoiding use of additives. Importantly, the method uses only ∼3 mg of tissue per sample and includes 12 heavy-amino-acid internal standards to enable quantification of the absolute levels of PBAAs with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples but is applicable to other plant tissues. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cppb.20084DOI Listing
December 2018

Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum.

Plant Physiol 2018 08 20;177(4):1425-1438. Epub 2018 Jun 20.

Department of Agronomy and Horticulture and Center for Plant Science Innovation, University of Nebraska, Lincoln, Nebraska 68588

Kafirins are the major storage proteins in sorghum () grains and form protein bodies with poor digestibility. Since kafirins are devoid of the essential amino acid lysine, they also impart poor protein quality to the kernel. The α-kafirins, which make up most of the total kafirins, are largely encoded by the family of highly similar genes. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to target the genes to create variants with reduced kafirin levels and improved protein quality and digestibility. A single guide RNA was designed to introduce mutations in a conserved region encoding the endoplasmic reticulum signal peptide of α-kafirins. Sequencing of kafirin PCR products revealed extensive edits in 25 of 26 events in one or multiple family members. T1 and T2 seeds showed reduced α-kafirin levels, and selected T2 events showed significantly increased grain protein digestibility and lysine content. Thus, a single consensus single guide RNA carrying target sequence mismatches is sufficient for extensive editing of all genes. The resulting quality improvements can be deployed rapidly for breeding and the generation of transgene-free, improved cultivars of sorghum, a major crop worldwide.
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http://dx.doi.org/10.1104/pp.18.00200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6084649PMC
August 2018

Topological Data Analysis as a Morphometric Method: Using Persistent Homology to Demarcate a Leaf Morphospace.

Front Plant Sci 2018 25;9:553. Epub 2018 Apr 25.

Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, United States.

Current morphometric methods that comprehensively measure shape cannot compare the disparate leaf shapes found in seed plants and are sensitive to processing artifacts. We explore the use of persistent homology, a topological method applied as a filtration across simplicial complexes (or more simply, a method to measure topological features of spaces across different spatial resolutions), to overcome these limitations. The described method isolates subsets of shape features and measures the spatial relationship of neighboring pixel densities in a shape. We apply the method to the analysis of 182,707 leaves, both published and unpublished, representing 141 plant families collected from 75 sites throughout the world. By measuring leaves from throughout the seed plants using persistent homology, a defined morphospace comparing all leaves is demarcated. Clear differences in shape between major phylogenetic groups are detected and estimates of leaf shape diversity within plant families are made. The approach predicts plant family above chance. The application of a persistent homology method, using topological features, to measure leaf shape allows for a unified morphometric framework to measure plant form, including shapes, textures, patterns, and branching architectures.
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http://dx.doi.org/10.3389/fpls.2018.00553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996898PMC
April 2018

Network-Guided GWAS Improves Identification of Genes Affecting Free Amino Acids.

Plant Physiol 2017 01 21;173(1):872-886. Epub 2016 Nov 21.

Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211 (R.A., A.B.).

Amino acids are essential for proper growth and development in plants. Amino acids serve as building blocks for proteins but also are important for responses to stress and the biosynthesis of numerous essential compounds. In seed, the pool of free amino acids (FAAs) also contributes to alternative energy, desiccation, and seed vigor; thus, manipulating FAA levels can significantly impact a seed's nutritional qualities. While genome-wide association studies (GWAS) on branched-chain amino acids have identified some regulatory genes controlling seed FAAs, the genetic regulation of FAA levels, composition, and homeostasis in seeds remains mostly unresolved. Hence, we performed GWAS on 18 FAAs from a 313-ecotype Arabidopsis (Arabidopsis thaliana) association panel. Specifically, GWAS was performed on 98 traits derived from known amino acid metabolic pathways (approach 1) and then on 92 traits generated from an unbiased correlation-based metabolic network analysis (approach 2), and the results were compared. The latter approach facilitated the discovery of additional novel metabolic interactions and single-nucleotide polymorphism-trait associations not identified by the former approach. The most prominent network-guided GWAS signal was for a histidine (His)-related trait in a region containing two genes: a cationic amino acid transporter (CAT4) and a polynucleotide phosphorylase resistant to inhibition with fosmidomycin. A reverse genetics approach confirmed CAT4 to be responsible for the natural variation of His-related traits across the association panel. Given that His is a semiessential amino acid and a potent metal chelator, CAT4 orthologs could be considered as candidate genes for seed quality biofortification in crop plants.
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http://dx.doi.org/10.1104/pp.16.01287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210728PMC
January 2017

ZEAXANTHIN EPOXIDASE Activity Potentiates Carotenoid Degradation in Maturing Seed.

Plant Physiol 2016 07 6;171(3):1837-51. Epub 2016 May 6.

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 (S.G.-J., P.M., M.M.-L., R.A., D.D.P.);Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA United Kingdom (S.G.-J., P.M.);Department of Crop Sciences, College of Agricultural, Consumer, and Environmental Sciences, University of Illinois, Urbana-Champaign, Illinois 61801 (A.E.L.);Division of Biological Sciences, University of Missouri, Columbia, Missouri 65201 (R.A.); andPlant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, New York 14853 (M.A.G.)

Elucidation of the carotenoid biosynthetic pathway has enabled altering the composition and content of carotenoids in various plants, but to achieve desired nutritional impacts, the genetic components regulating carotenoid homeostasis in seed, the plant organ consumed in greatest abundance, must be elucidated. We used a combination of linkage mapping, genome-wide association studies (GWAS), and pathway-level analysis to identify nine loci that impact the natural variation of seed carotenoids in Arabidopsis (Arabidopsis thaliana). ZEAXANTHIN EPOXIDASE (ZEP) was the major contributor to carotenoid composition, with mutants lacking ZEP activity showing a remarkable 6-fold increase in total seed carotenoids relative to the wild type. Natural variation in ZEP gene expression during seed development was identified as the underlying mechanism for fine-tuning carotenoid composition, stability, and ultimately content in Arabidopsis seed. We previously showed that two CAROTENOID CLEAVAGE DIOXYGENASE enzymes, CCD1 and CCD4, are the primary mediators of seed carotenoid degradation, and here we demonstrate that ZEP acts as an upstream control point of carotenoid homeostasis, with ZEP-mediated epoxidation targeting carotenoids for degradation by CCD enzymes. Finally, four of the nine loci/enzymatic activities identified as underlying natural variation in Arabidopsis seed carotenoids also were identified in a recent GWAS of maize (Zea mays) kernel carotenoid variation. This first comparison of the natural variation in seed carotenoids in monocots and dicots suggests a surprising overlap in the genetic architecture of these traits between the two lineages and provides a list of likely candidates to target for selecting seed carotenoid variation in other species.
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http://dx.doi.org/10.1104/pp.16.00604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936585PMC
July 2016

The role of photosynthesis and amino acid metabolism in the energy status during seed development.

Front Plant Sci 2014 3;5:447. Epub 2014 Sep 3.

Max-Planck-Institut für Molekulare Pflanzenphysiologie Potsdam-Golm, Germany.

Seeds are the major organs responsible for the evolutionary upkeep of angiosperm plants. Seeds accumulate significant amounts of storage compounds used as nutrients and energy reserves during the initial stages of seed germination. The accumulation of storage compounds requires significant amounts of energy, the generation of which can be limited due to reduced penetration of oxygen and light particularly into the inner parts of seeds. In this review, we discuss the adjustment of seed metabolism to limited energy production resulting from the suboptimal penetration of oxygen into the seed tissues. We also discuss the role of photosynthesis during seed development and its contribution to the energy status of developing seeds. Finally, we describe the contribution of amino acid metabolism to the seed energy status, focusing on the Asp-family pathway that leads to the synthesis and catabolism of Lys, Thr, Met, and Ile.
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http://dx.doi.org/10.3389/fpls.2014.00447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4153028PMC
September 2014

Carotenoid cleavage dioxygenase4 is a negative regulator of β-carotene content in Arabidopsis seeds.

Plant Cell 2013 Dec 24;25(12):4812-26. Epub 2013 Dec 24.

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.

Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed β-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for β-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID cleavage dioxygenase4 (CCD4) as a major negative regulator of seed carotenoid content, especially β-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing β-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with β-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in β-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops.
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http://dx.doi.org/10.1105/tpc.113.119677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903989PMC
December 2013

Genome-wide analysis of branched-chain amino acid levels in Arabidopsis seeds.

Plant Cell 2013 Dec 24;25(12):4827-43. Epub 2013 Dec 24.

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.

Branched-chain amino acids (BCAAs) are three of the nine essential amino acids in human and animal diets and are important for numerous processes in development and growth. However, seed BCAA levels in major crops are insufficient to meet dietary requirements, making genetic improvement for increased and balanced seed BCAAs an important nutritional target. Addressing this issue requires a better understanding of the genetics underlying seed BCAA content and composition. Here, a genome-wide association study and haplotype analysis for seed BCAA traits in Arabidopsis thaliana revealed a strong association with a chromosomal interval containing two branched-chain amino acid transferases, BCAT1 and BCAT2. Linkage analysis, reverse genetic approaches, and molecular complementation analysis demonstrated that allelic variation at BCAT2 is responsible for the natural variation of seed BCAAs in this interval. Complementation analysis of a bcat2 null mutant with two significantly different alleles from accessions Bayreuth-0 and Shahdara is consistent with BCAT2 contributing to natural variation in BCAA levels, glutamate recycling, and free amino acid homeostasis in seeds in an allele-dependent manner. The seed-specific phenotype of bcat2 null alleles, its strong transcription induction during late seed development, and its subcellular localization to the mitochondria are consistent with a unique, catabolic role for BCAT2 in BCAA metabolism in seeds.
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http://dx.doi.org/10.1105/tpc.113.119370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903990PMC
December 2013

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.

Plant Physiol 2013 Feb 18;161(2):628-43. Epub 2012 Dec 18.

Max-Planck-Institut für Molekulare Pflanzenphysiologie, 14476 Potsdam-Golm, Germany.

The aim of this work was to investigate the effect of decreased cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and plastidic NADP-dependent malic enzyme (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 on 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 transformants, which suggests that it is due to an altered cellular redox status. Moreover, metabolic profiling and feeding experiments involving positionally labeled glucoses of fruits lacking in plastidic NADP-ME 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 an excess of oxaloacetate is converted to aspartate and reintroduced in the TCA cycle via 2-oxoglutarate/glutamate. On the other hand, the plastidic NADP-ME antisense lines were characterized by no changes in respiration rates and TCA cycle flux, which together with increases of pyruvate kinase and phosphoenolpyruvate carboxylase activities indicate that pyruvate is supplied 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.
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http://dx.doi.org/10.1104/pp.112.211094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561009PMC
February 2013

Deciphering energy-associated gene networks operating in the response of Arabidopsis plants to stress and nutritional cues.

Plant J 2012 Jun 4;70(6):954-66. Epub 2012 Apr 4.

Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel.

Plants need to continuously adjust their transcriptome in response to various stresses that lead to inhibition of photosynthesis and the deprivation of cellular energy. This adjustment is triggered in part by a coordinated re-programming of the energy-associated transcriptome to slow down photosynthesis and activate other energy-promoting gene networks. Therefore, understanding the stress-related transcriptional networks of genes belonging to energy-associated pathways is of major importance for engineering stress tolerance. In a bioinformatics approach developed by our group, termed 'gene coordination', we previously divided genes encoding for enzymes and transcription factors in Arabidopsis thaliana into three clusters, displaying altered coordinated transcriptional behaviors in response to multiple biotic and abiotic stresses (Plant Cell, 23, 2011, 1264). Enrichment analysis indicated further that genes controlling energy-associated metabolism operate as a compound network in response to stress. In the present paper, we describe in detail the network association of genes belonging to six central energy-associated pathways in each of these three clusters described in our previous paper. Our results expose extensive stress-associated intra- and inter-pathway interactions between genes from these pathways, indicating that genes encoding proteins involved in energy-associated metabolism are expressed in a highly coordinated manner. We also provide examples showing that this approach can be further utilized to elucidate candidate genes for stress tolerance and functions of isozymes.
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http://dx.doi.org/10.1111/j.1365-313X.2012.04926.xDOI Listing
June 2012

Targeted enhancement of glutamate-to-γ-aminobutyrate conversion in Arabidopsis seeds affects carbon-nitrogen balance and storage reserves in a development-dependent manner.

Plant Physiol 2011 Nov 15;157(3):1026-42. Epub 2011 Sep 15.

French Associates Institute for Biotechnology and Agriculture of Dryland, Blaustein Institutes for Desert Research, Ben-Gurion University of Negev, Midreshet Ben Gurion 84990, Israel.

In seeds, glutamate decarboxylase (GAD) operates at the metabolic nexus between carbon and nitrogen metabolism by catalyzing the unidirectional decarboxylation of glutamate to form γ-aminobutyric acid (GABA). To elucidate the regulatory role of GAD in seed development, we generated Arabidopsis (Arabidopsis thaliana) transgenic plants expressing a truncated GAD from Petunia hybrida missing the carboxyl-terminal regulatory Ca(2+)-calmodulin-binding domain under the transcriptional regulation of the seed maturation-specific phaseolin promoter. Dry seeds of the transgenic plants accumulated considerable amounts of GABA, and during desiccation the content of several amino acids increased, although not glutamate or proline. Dry transgenic seeds had higher protein content than wild-type seeds but lower amounts of the intermediates of glycolysis, glycerol and malate. The total fatty acid content of the transgenic seeds was 50% lower than in the wild type, while acyl-coenzyme A accumulated in the transgenic seeds. Labeling experiments revealed altered levels of respiration in the transgenic seeds, and fractionation studies indicated reduced incorporation of label in the sugar and lipid fractions extracted from transgenic seeds. Comparative transcript profiling of the dry seeds supported the metabolic data. Cellular processes up-regulated at the transcript level included the tricarboxylic acid cycle, fatty acid elongation, the shikimate pathway, tryptophan metabolism, nitrogen-carbon remobilization, and programmed cell death. Genes involved in the regulation of germination were similarly up-regulated. Taken together, these results indicate that the GAD-mediated conversion of glutamate to GABA during seed development plays an important role in balancing carbon and nitrogen metabolism and in storage reserve accumulation.
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http://dx.doi.org/10.1104/pp.111.179986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3252140PMC
November 2011

A friend in need is a friend indeed: understanding stress-associated transcriptional networks of plant metabolism using cliques of coordinately expressed genes.

Plant Signal Behav 2011 Sep 17;6(9):1294-6. Epub 2011 Aug 17.

Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel.

The response of plants to environmental cues, particularly stresses, involves the coordinated induction or repression of gene expression. In a previous study, we developed a bioinformatics approach to analyze the mutual expression pattern of genes encoding transcription factors and metabolic enzymes upon exposure of Arabidopsis plants to abiotic and biotic stresses. The analysis resulted in three gene clusters, each displaying a unique expression pattern. In the present addendum, we address the composition of each of these three clusters in regard to the functional identity of their encoded proteins as enzymes or transcription factors.
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http://dx.doi.org/10.4161/psb.6.9.16567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258055PMC
September 2011

Coordinated gene networks regulating Arabidopsis plant metabolism in response to various stresses and nutritional cues.

Plant Cell 2011 Apr 12;23(4):1264-71. Epub 2011 Apr 12.

Department of Plant Sciences, Weizman Institute of Science, Rehovot 76100, Israel.

The expression pattern of any pair of genes may be negatively correlated, positively correlated, or not correlated at all in response to different stresses and even different progression stages of the stress. This makes it difficult to identify such relationships by classical statistical tools such as the Pearson correlation coefficient. Hence, dedicated bioinformatics approaches that are able to identify groups of cues in which there is a positive or negative expression correlation between pairs or groups of genes are called for. We herein introduce and discuss a bioinformatics approach, termed Gene Coordination, that is devoted to the identification of specific or multiple cues in which there is a positive or negative coordination between pairs of genes and can further incorporate additional coordinated genes to form large coordinated gene networks. We demonstrate the utility of this approach by providing a case study in which we were able to discover distinct expression behavior of the energy-associated gene network in response to distinct biotic and abiotic stresses. This bioinformatics approach is suitable to a broad range of studies that compare treatments versus controls, such as effects of various cues, or expression changes between a mutant and the control wild-type genotype.
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http://dx.doi.org/10.1105/tpc.110.082867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3101534PMC
April 2011

A seed high-lysine trait is negatively associated with the TCA cycle and slows down Arabidopsis seed germination.

New Phytol 2011 Jan 11;189(1):148-59. Epub 2010 Oct 11.

Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel.

• Lysine is a nutritionally important essential amino acid, but significant elevation of its levels in Arabidopsis seeds, by enhancing its synthesis and blocking its catabolism, causes a retardation of germination. Here, we hypothesized that this negative effect is associated with changes in primary metabolism and gene expression programs that are essential for early germination. • Seeds at different stages of germination sensu stricto of the seed-high-lysine genotype were subjected to detailed analysis of primary metabolism, using GC-MS, as well as microarray analysis and two-dimensional, isoelectric focusing, sodium dodecylsulfate polyacrylamide gel electrophoresis, to detect storage protein mobilization. • Our results exposed a major negative effect of the seed-specific increased lysine synthesis and knockout of its catabolism on the levels of a number of TCA cycle metabolites. This metabolic alteration also influences significantly the transcriptome, primarily attenuating the boost of specific transcriptional programs that are essential for seedling establishment, such as the onset of photosynthesis, as well as the turnover of specific transcriptional programs associated with seed embryonic traits. • Our results indicate that catabolism of the aspartic acid family of amino acids is an important contributor to the energy status of plants, and hence to the onset of autotrophic growth-associated processes during germination.
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http://dx.doi.org/10.1111/j.1469-8137.2010.03478.xDOI Listing
January 2011

Principal transcriptional regulation and genome-wide system interactions of the Asp-family and aromatic amino acid networks of amino acid metabolism in plants.

Amino Acids 2010 Oct 4;39(4):1023-8. Epub 2010 Apr 4.

Department of Plant Sciences, The Weizmann Institute of Science, Rehovot, Israel.

Amino acid metabolism is among the most important and best recognized networks within biological systems. In plants, amino acids serve multiple functions associated with growth. Besides their function in protein synthesis, the amino acids are also catabolized into energy-associated metabolites as well we into numerous secondary metabolites, which are essential for plant growth and response to various stresses. Despite the central importance of amino acids in plants growth, elucidation of the regulation of amino acid metabolism within the context of the entire system, particularly transcriptional regulation, is still in its infancy. The different amino acids are synthesized by a number of distinct metabolic networks, which are expected to possess regulatory cross interactions between them for proper coordination of their interactive functions, such as incorporation into proteins. Yet, individual amino acid metabolic networks are also expected to differentially cross interact with various genome-wide gene expression programs and metabolic networks, in respect to their functions as precursors for various metabolites with distinct functions. In the present review, we discuss our recent genomics, metabolic and bioinformatics studies, which were aimed at addressing these questions, focusing mainly on the Asp-family metabolic network as the main example and also comparing it to the aromatic amino acids metabolic network as a second example (Angelovici et al. in Plant Physiol 151:2058-2072, 2009; Less and Galili in BMC Syst Biol 3:14, 2009; Tzin et al. in Plant J 60:156-167, 2009). Our focus on these two networks is because of the followings: (i) both networks are central to plant metabolism and growth and are also precursors for a wide range of primary and secondary metabolites that are indispensable to plant growth; (ii) the amino acids produced by these two networks are also essential to the nutrition and health of human and farm animals; and (iii) both networks contain branched pathways requiring extensive regulation of fluxes between the different branches. Additional views on the biochemistry, regulation and functional significance of the Asp-family and aromatic amino acid networks and some of their associated metabolites that are discussed in the present report, as well as the nutritional importance of Lys and Trp to human and farm animals, and attempts to improve Lys level in crop plants, can be obtained from the following reviews as examples (Radwanski and Last in Plant Cell 7:921-934, 1995; Halkier and Gershenzon in Annu Rev Plant Biol 57:303-333, 2006; Ufaz and Galili in Plant Physiol 147:954-961, 2008; Jander and Joshi in Mol Plant 3:54-65, 2010).
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http://dx.doi.org/10.1007/s00726-010-0566-7DOI Listing
October 2010

Seed desiccation: a bridge between maturation and germination.

Trends Plant Sci 2010 Apr;15(4):211-8

Department of Plant Science, the Weizmann Institute of Science, Rehovot, Israel.

The development of orthodox seeds concludes by a desiccation phase. The dry seeds then enter a phase of dormancy, also called the after-ripening phase, and become competent for germination. We discuss physiological processes as well as gene expression and metabolic programs occurring during the desiccation phase in respect to their contribution to the desiccation tolerance, dormancy competence and successful germination of the dry seeds. The transition of developing seeds from the phase of reserve accumulation to desiccation is associated with distinct gene expression and metabolic switches. Interestingly, a significant proportion of the gene expression and metabolic signatures of seed desiccation resemble those characterizing seed germination, implying that the preparation of the seeds for germination begins already during seed desiccation.
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http://dx.doi.org/10.1016/j.tplants.2010.01.003DOI Listing
April 2010

Deciphering transcriptional and metabolic networks associated with lysine metabolism during Arabidopsis seed development.

Plant Physiol 2009 Dec 25;151(4):2058-72. Epub 2009 Sep 25.

Department of Plant Sciences, Weizmann Institute of Science, 76100 Rehovot, Israel.

In order to elucidate transcriptional and metabolic networks associated with lysine (Lys) metabolism, we utilized developing Arabidopsis (Arabidopsis thaliana) seeds as a system in which Lys synthesis could be stimulated developmentally without application of chemicals and coupled this to a T-DNA insertion knockout mutation impaired in Lys catabolism. This seed-specific metabolic perturbation stimulated Lys accumulation starting from the initiation of storage reserve accumulation. Our results revealed that the response of seed metabolism to the inducible alteration of Lys metabolism was relatively minor; however, that which was observable operated in a modular manner. They also demonstrated that Lys metabolism is strongly associated with the operation of the tricarboxylic acid cycle while largely disconnected from other metabolic networks. In contrast, the inducible alteration of Lys metabolism was strongly associated with gene networks, stimulating the expression of hundreds of genes controlling anabolic processes that are associated with plant performance and vigor while suppressing a small number of genes associated with plant stress interactions. The most pronounced effect of the developmentally inducible alteration of Lys metabolism was an induction of expression of a large set of genes encoding ribosomal proteins as well as genes encoding translation initiation and elongation factors, all of which are associated with protein synthesis. With respect to metabolic regulation, the inducible alteration of Lys metabolism was primarily associated with altered expression of genes belonging to networks of amino acids and sugar metabolism. The combined data are discussed within the context of network interactions both between and within metabolic and transcriptional control systems.
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http://dx.doi.org/10.1104/pp.109.145631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785976PMC
December 2009

Arabidopsis seed development and germination is associated with temporally distinct metabolic switches.

Plant Physiol 2006 Nov 8;142(3):839-54. Epub 2006 Sep 8.

Department of Plant Sciences, the Weizmann Institute of Science, 76100 Rehovot, Israel.

While the metabolic networks in developing seeds during the period of reserve accumulation have been extensively characterized, much less is known about those present during seed desiccation and subsequent germination. Here we utilized metabolite profiling, in conjunction with selective mRNA and physiological profiling to characterize Arabidopsis (Arabidopsis thaliana) seeds throughout development and germination. Seed maturation was associated with a significant reduction of most sugars, organic acids, and amino acids, suggesting their efficient incorporation into storage reserves. The transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids, and shikimate-derived metabolites. In contrast, seed vernalization was associated with a decrease in the content of several of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly, the levels of other metabolites significantly increased during vernalization and were boosted further during germination sensu stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as determined by semiquantitative reverse transcription-polymerase chain reaction and analysis of publicly available microarray data. When taken together our results provide a comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct metabolic switches during vernalization and early germination.
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http://dx.doi.org/10.1104/pp.106.086694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1630763PMC
November 2006
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