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.

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.

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.

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.

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.

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.

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).

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

Kafirins are the major storage proteins in sorghum (Sorghum bicolor) 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 k1C 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 k1C 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 k1C 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 k1C 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.

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

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.

ZEAXANTHIN EPOXIDASE Activity Potentiates Carotenoid Degradation in Maturing Seed.

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.

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

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.

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

Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed ß-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 ß-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 ß-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing ß-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 ß-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in ß-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.

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 (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.

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

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.

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.

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

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.

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.