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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 

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. 

SUMMARY Extracellular ATP (eATP) is known to act as a danger signal in both plants and animals. In plants, eATP is recognized by the plasma membrane (PM)‐localized receptor P2K1 (LecRK‐I.9). Among the first measurable responses to eATP addition is a rapid rise in cytoplasmic free calcium levels ([Ca²⁺]_cyt), which requires P2K1. However, the specific transporter/channel proteins that mediate this rise in [Ca²⁺]_cytare unknown. Through a forward genetic screen, we identified an Arabidopsis ethylmethanesulfonate (EMS) mutant impaired in the [Ca²⁺]_cytresponse to eATP. Positional cloning revealed that the mutation resided in thecngc6gene, which encodes cyclic nucleotide‐gated ion channel 6 (CNGC6). Mutation of theCNGC6gene led to a notable decrease in the PM inward Ca²⁺current in response to eATP. eATP‐induced mitogen‐activated protein kinase activation and gene expression were also significantly lower incngc6mutant plants. In addition,cngc6mutant plants were also more susceptible to the bacterial pathogenPseudomonas syringae. Taken together, our results indicate that CNGC6 plays a crucial role in mediating eATP‐induced [Ca²⁺]_cytsignaling, as well as plant immunity.