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Abstract In plants, embryo size is determined via interactions between metabolic and developmental signals. Maize (Zea mays) big embryo 6 (bige6) enhances embryo size while sharply reducing plant growth. Here, we show that BigE6 encodes a plastidial prephenate aminotransferase (PPA-AT), a key enzyme in the arogenate pathway for L-phenylalanine (Phe) and L-tyrosine (Tyr) biosynthesis. The maize BigE6 paralog, BigE6Like, encodes a cytosol-localized PPA-AT, revealing Phe and Tyr biosynthesis via cytosolic arogenate as a potential alternative to the known cytosolic phenylpyruvate pathway. Moreover, the single PPA-AT gene of Arabidopsis (Arabidopsis thaliana) encodes plastidial and cytosolic enzymes by alternative splicing. Transgenic rescue of a ppa-at mutant in Arabidopsis demonstrates that the plastidial PPA-AT is indispensable for seed formation due, in part, to its essential role in the female gametophyte. Leaves of bige6 maize maintained overall homeostasis for aromatic amino acids and downstream metabolites, revealing a resilience of mechanisms that scale growth to a limiting supply of Phe and Tyr. In bige6 seeds, broad perturbation of amino acid homeostasis is associated with transcriptomic upregulation of growth processes in the embryo and endosperm, implicating amino acid signaling in the regulation of embryo size. Our findings reveal the complexity and developmental dependence of growth responses to limiting amino acid biosynthesis. 

Abstract Translation of mRNA into functional proteins is a fundamental process underlying many aspects of plant growth and development. Yet, the role of translational regulation in plants across diverse tissue types, including seeds, is not well known due to the lack of methods targeting these processes. Studying the seed translatome could unveil seed‐specific regulatory mechanisms, offering valuable insights for breeding efforts to enhance seed traits. Polysome profiling is a widely used technique for studying mRNAs being translated. However, the traditional method is time‐consuming and has a low polysome recovery rate; therefore, it requires substantial starting material. This is particularly challenging for species or mutants with limited seed quantities. Additionally, seed polysome fractions often yield low quality RNA due to the abundance of various compounds that interfere with conventional RNA extraction protocols. Here we present a robust polysome extraction method incorporating a size‐exclusion step for polysome concentration streamlined with a rapid RNA extraction method optimized for seeds. This protocol works across multiple plant species and offers increased speed and robustness, requiring less than half the amount of seed tissue and time compared to conventional methods while ensuring high polysome recovery and yield of high‐quality RNA for downstream experiments. These features make this protocol an ideal tool for studying seed translation efficiency and hold broad applicability across various plant species and tissues. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Robust polysome extraction for seeds Basic Protocol 2: Rapid fraction total RNA extraction 

The titan arum (Amorphophallus titanum), commonly known as the corpse flower, produces the largest unbranched inflorescence in the world. Its rare blooms last only a few days and are notable both for their burst of thermogenic activity and for the odor of rotting flesh by which they attract pollinators. Studies on the titan arum can therefor lend insight into the mechanisms underlying thermogenesis as well as the production of sulfur-based volatiles, about which little is known in plants. Here, we made use of transcriptome and metabolite analyses to uncover underlying mechanisms that enable thermogenesis and volatile production in the titan arum. The ability to perform thermogenesis correlated with the expression of genes involved in bypass steps for the mitochondrial electron transport chain, in particular alternative oxidase expression, and through our analysis is placed within the context of sugar transport and metabolism. The major odorants produced by the titan arum are dimethyl disulfide and dimethyl trisulfide, and we identified pathways for sulfur transport and metabolism that culminate in the production of methionine, which our analysis identifies as the amino acid substrate for production of these odorants. Putrescine, derived from arginine, was identified as an additional and previously unrecognized component of the titan arum's odor. Levels of free methionine and putrescine were rapidly depleted during thermogenesis, consistent with roles in production of the titan arum's odor. Models for how tissues of the titan arum contribute to thermogenesis and volatile production are proposed. 

Abstract PremiseThe 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 usedCakile 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. MethodsUsing a combination of ionomics, free amino acid profiling, and comparative genomics, we characterize aspects of salt stress response inC. maritimaand identify retained duplicate genes that have likely enabled adaptation to salt and mild levels of cadmium. ResultsCakile maritimais 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 inC. maritimaand 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 inC. maritimaabiotic stress tolerance. ConclusionsThese 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. 

Abstract 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 

Abstract 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 

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 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. 

Abstract 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.