Halophytes and heavy metals: A multi-omics approach to understand the role of gene and genome duplication in the abiotic stress tolerance of Cakile maritima.

PREMISE: The origin of diversity is a fundamental biological question. Gene duplications are one mechanism that provides raw material for the emergence of novel traits, but evolutionary outcomes depend on which genes are retained and how they become functionalized. Yet, following different duplication types (polyploidy and tandem duplication), the events driving gene retention and functionalization remain poorly understood. Here we used Cakile maritima, a species that is tolerant to salt and heavy metals and shares an ancient whole-genome triplication with closely related salt-sensitive mustard crops (Brassica), as a model to explore the evolution of abiotic stress tolerance following polyploidy. METHODS: Using a combination of ionomics, free amino acid profiling, and comparative genomics, we characterize aspects of salt stress response in C. maritima and identify retained duplicate genes that have likely enabled adaptation to salt and mild levels of cadmium. RESULTS: Cakile maritima is tolerant to both cadmium and salt treatments through uptake of cadmium in the roots. Proline constitutes greater than 30% of the free amino acid pool in C. maritima and likely contributes to abiotic stress tolerance. We find duplicated gene families are enriched in metabolic and transport processes and identify key transport genes that may be involved in C. maritima abiotic stress tolerance. CONCLUSIONS: These findings identify pathways and genes that could be used to enhance plant resilience and provide a putative understanding of the roles of duplication types and retention on the evolution of abiotic stress response.

Reducing Acrylamide Formation Potential by Targeting Free Asparagine Accumulation in Seeds.

Acrylamide is a probable carcinogen in humans and is formed when reducing sugars react with free asparagine (Asn) during thermal processing of food. Although breeding for low reducing sugars worked well in potatoes, it is less successful in cereals. However, reducing free Asn in cereals has great potential for reducing acrylamide formation, despite the role that Asn plays in nitrogen transport and amino acid biosynthesis. In this perspective, we summarize the efforts aimed at reducing free Asn in cereal grains and discuss the potentials and challenges associated with targeting this essential amino acid, especially in a seed-specific manner.

Editing the 19 kDa alpha-zein gene family generates non-opaque2-based quality protein maize.

Maize grain is deficient in lysine. While the opaque2 mutation increases grain lysine, o2 is a transcription factor that regulates a wide network of genes beyond zeins, which leads to pleiotropic and often negative effects. Additionally, the drastic reduction in 19 kDa and 22 kDa alpha-zeins causes a floury kernel, unsuitable for agricultural use. Quality protein maize (QPM) overcame the undesirable kernel texture through the introgression of modifying alleles. However, QPM still lacks a functional o2 transcription factor, which has a penalty on non-lysine amino acids due to the o2 mutation. CRISPR/cas9 gives researchers the ability to directly target genes of interest. In this paper, gene editing was used to specifically target the 19 kDa alpha zein gene family. This allows for proteome rebalancing to occur without an o2 mutation and without a total alpha-zein knockout. The results showed that editing some, but not all, of the 19 kDa zeins resulted in up to 30% more lysine. An edited line displayed an increase of 30% over the wild type. While not quite the 55% lysine increase displayed by QPM, the line had little collateral impact on other amino acid levels compared to QPM. Additionally, the edited line containing a partially reduced 19 kDa showed an advantage in kernel texture that had a complete 19 kDa knockout. These results serve as proof of concept that editing the 19 kDa alpha-zein family alone can enhance lysine while retaining vitreous endosperm and a functional O2 transcription factor.

A High-Throughput Absolute Quantification of Protein-Bound Sulfur Amino Acids from Model and Crop Plant Seeds.

In this procedure, we describe a high-throughput absolute quantification protocol for the protein-bound sulfur amino acids, cysteine (Cys) and methionine (Met), from plant seeds. This procedure consists of performic acid oxidation that transforms bound Cys into cysteic acid (CysA) and bound Met into methionine sulfone (MetS) followed by acid hydrolysis. The absolute quantification step is performed by multiple reaction monitoring tandem mass spectrometry (LC-MS/MS). The approach facilitates the analysis of a few hundred samples per week by using a 96-well plate extraction setup. Importantly, the method uses only ∼4 mg of tissue per sample and uses the common acid hydrolysis protocol, followed by water extraction that includes DL-Ser-d3 and L-Met-d3 as internal standards to enable the quantification of the absolute levels of the protein-bound Cys and Met with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples from Arabidopsis thaliana, Glycine max, and Zea mays but could be applied to other plant tissues. © 2023 Wiley Periodicals LLC. Basic Protocol: Analysis of protein-bound cysteine and methionine from seeds.

A High-Throughput Absolute Quantification of Protein-Bound Tryptophan from Model and Crop Seeds.

This protocol describes a high-throughput absolute quantification protocol for the aromatic essential amino acid, tryptophan (Trp). This procedure consists of a milligram-scale alkaline hydrolysis followed by an absolute quantification step using a multiple reaction monitoring tandem mass spectrometric (LC-MS/MS) detection method. The approach facilitates the analysis of a few hundred samples per week by using a 96-well plate extraction setup. Importantly, the method uses only ∼4 mg of tissue per sample and uses the common alkaline hydrolysis protocol, followed by water extraction that includes L-Trp-d5 as an internal standard to enable the quantification of the absolute level of the bound Trp with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples for Arabidopsis thaliana, Glycine max, and Zea mays but could be applied to other plant tissues. © 2023 Wiley Periodicals LLC. Basic Protocol: Analysis of protein-bound tryptophan from seeds.

Large and stable genome edits at the sorghum alpha kafirin locus result in changes in chromatin accessibility and globally increased expression of genes encoding lysine enrichment.

Introduction: Sorghum is a resilient and widely cultivated grain crop used for feed and food. However, it's grain is deficient in lysine, an essential amino acid. This is due to the primary seed storage proteins, the alpha-kafirins, lacking lysine. It has been observed that reductions in alpha-kafirin protein results in rebalancing of the seed proteome and a corresponding increase in non-kafirin proteins which leads to an increased lysine content. However, the mechanisms underlying proteome rebalancing are unclear. This study characterizes a previously developed gene edited sorghum line, with deletions at the alpha kafirin locus. Methods: A single consensus guide RNA leads to tandem deletion of multiple members of the gene family in addition to the small target site mutations in remaining genes. RNA-seq and ATAC-seq were utilized to identify changes in gene expression and chromatin accessibility in developing kernels in the absence of most alpha-kafirin expression. Results: Several differentially accessible chromatin regions and differentially expressed genes were identified. Additionally, several genes upregulated in the edited sorghum line were common with their syntenic orthologues differentially expressed in maize prolamin mutants. ATAC-seq showed enrichment of the binding motif for ZmOPAQUE 11, perhaps indicating the transcription factor's involvement in the kernel response to reduced prolamins. Discussion: Overall, this study provides a resource of genes and chromosomal regions which may be involved in sorghum's response to reduced seed storage proteins and the process of proteome rebalancing.

A plant balancing act: Meshing new and existing metabolic pathways towards an optimized system.

Specialized metabolic pathways evolve from existing pathways, creating new functionality potentially boosting fitness. However, how these pathways are integrated into a pre-existing working and well-balanced metabolic system is unclear. They could be integrated to the system as a functional appendage, or they could be fully embedded into primary metabolism by establishing new biochemical and regulatory connections. A full integration into the primary metabolic system requires substantial system re-wiring and because of this complexity, the latter is often not experimentally pursued. New studies provide evidence that some specialized metabolic pathways are fully embedded in primary metabolism with extensive new regulatory and biochemical connections. This suggests, that we should consider whether other specialized metabolic pathways could be fully integrated rather than being simple appendages. In this mini review, we survey compelling evidence supporting that some specialized metabolic pathways are fully integrated and ask if these metabolites now act as de-facto primary metabolites?

Multiomics approach reveals a role of translational machinery in shaping maize kernel amino acid composition.

Maize (Zea mays) seeds are a good source of protein, despite being deficient in several essential amino acids. However, eliminating the highly abundant but poorly balanced seed storage proteins has revealed that the regulation of seed amino acids is complex and does not rely on only a handful of proteins. In this study, we used two complementary omics-based approaches to shed light on the genes and biological processes that underlie the regulation of seed amino acid composition. We first conducted a genome-wide association study to identify candidate genes involved in the natural variation of seed protein-bound amino acids. We then used weighted gene correlation network analysis to associate protein expression with seed amino acid composition dynamics during kernel development and maturation. We found that almost half of the proteome was significantly reduced during kernel development and maturation, including several translational machinery components such as ribosomal proteins, which strongly suggests translational reprogramming. The reduction was significantly associated with a decrease in several amino acids, including lysine and methionine, pointing to their role in shaping the seed amino acid composition. When we compared the candidate gene lists generated from both approaches, we found a nonrandom overlap of 80 genes. A functional analysis of these genes showed a tight interconnected cluster dominated by translational machinery genes, especially ribosomal proteins, further supporting the role of translation dynamics in shaping seed amino acid composition. These findings strongly suggest that seed biofortification strategies that target the translation machinery dynamics should be considered and explored further.

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

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 Arabidopsis thaliana 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.

Since plants cannot move, they have evolved chemical defenses to help them respond to changes in their surroundings. For example, where animals run from predators, plants may produce toxins to put predators off. This approach is why plants are such a rich source of drugs, poisons, dyes and other useful substances. The chemicals plants produce are known as specialized metabolites, and they can change a lot between, and even within, plant species. The variety of specialized metabolites is a result of genetic changes and evolution over millions of years. Evolution is a slow process, yet plants are able to rapidly develop new specialized metabolites to protect them from new threats. Even different populations of the same species produce many distinct metabolites that help them survive in their surroundings. However, the factors that lead plants to produce new metabolites are not well understood, and it is not known how this affects genetic variation. To gain a better understanding of this process, Katz et al. studied 797 European variants of a common weed species called Arabidopsis thaliana, which is widely studied. The investigation found that many factors affect the range of specialized metabolites in each variant. These included local geography and environment, as well as genetics and population history (demography). Katz et al. revealed a pattern of relationships between the variants that could mirror their evolutionary history as the species spread and adapted to new locations. These results highlight the complex network of factors that affect plant evolution. Rapid diversification is key to plant survival in new and changing environments and has resulted in a wide range of specialized metabolites. As such they are of interest both for studying plant evolution and for understanding their ecology. Expanding similar work to more populations and other species will broaden the scope of our ability to understand how plants adapt to their surroundings.

Final Selection of Quality Protein Popcorn Hybrids.

Quality Protein Popcorn (QPP) BC2F5 inbred lines were produced through an interpopulation breeding system between Quality Protein Maize dent (QPM) and elite popcorn germplasm. In 2019, five QPP F1 hybrids were selected for further evaluation due to superior agronomics, endosperm protein quality, and popping quality traits. Though these BC2F5 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, BC2F5 inbred lines were crossed to their popcorn parents and BC3F4 inbred lines were produced for hybridization to test the agronomic, protein, and popping trait effects from an additional QPP by popcorn backcross. Second, BC2- and BC3-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 BC3 cultivars compared to their BC2 counterparts, and yield averages were significantly lower in BC3-derived QPP hybrids compared to the BC2 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 BC2F5 population and three from the BC3F4 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.

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

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 Arabidopsis thaliana 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.

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

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 opaque-2 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.

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

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.

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

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.

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

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 (Arabidopsis thaliana) 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.

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

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).

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

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.

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

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).