Upon storage, seeds inevitably age and lose their viability over time, which determines their longevity. Longevity correlates with successful seed germination and enhancing this trait is of fundamental importance for long-term seed storage (germplasm conservation) and crop improvement. Seed longevity is governed by a complex interplay between genetic factors and environmental conditions experienced during seed development and after-ripening that will shape seed physiology. Several factors have been associated with seed ageing such as oxidative stress responses, DNA repair enzymes, and composition of seed layers. Phytohormones, mainly abscisic acid, auxins, and gibberellins, have also emerged as prominent endogenous regulators of seed longevity, and their study has provided new regulators of longevity. Gaining a thorough understanding of how hormonal signalling genes and pathways are integrated with downstream mechanisms related to seed longevity is essential for formulating strategies aimed at preserving seed quality and viability. A relevant aspect related to research in seed longevity is the existence of significant differences between results depending on the seed equilibrium relative humidity conditions used to study seed ageing. Hence, this review delves into the genetic, environmental and experimental factors affecting seed ageing and longevity, with a particular focus on their hormonal regulation. We also provide gene network models underlying hormone signalling aimed to help visualize their integration into seed longevity and ageing. We believe that the format used to present the information bolsters its value as a resource to support seed longevity research for seed conservation and crop improvement.

The two most abundant macronutrients in plant cells are carbon (C) and nitrogen (N). Coordination of their cellular metabolism is a fundamental factor in guaranteeing the optimal growth and development of plants. N availability and assimilation profoundly affect plant gene expression and modulate root and stem architecture, thus affecting whole plant growth and crop yield. N status also affects C fixation, as it is an important component of the photosynthetic machinery in leaves. Reciprocally, increasing C supply promotes N uptake and assimilation. There is extensive knowledge of the different mechanisms that plants use for sensing and signalling their nutritional status to regulate the assimilation, metabolism and transport of C and N. However, the crosstalk between C and N pathways has received much less attention. Plant growth and development are greatly affected by suboptimal C/N balance, which can arise from nutrient deficiencies or/and environmental cues. Mechanisms that integrate and respond to changes in this specific nutritional balance have started to arise. This review will examine the specific responses to C/N imbalance in plants by focusing on the main inorganic and organic metabolites involved, how they are sensed and transported, and the interconnection between the early signalling components and hormonal networks that underlies plants’ adaptive responses.

In a microgravity environment, without any gravitropic signal, plants are not able to define and establish a longitudinal growth axis. Consequently, absorption of water and nutrients by the root and exposure of leaves to sunlight for efficient photosynthesis is hindered. In these conditions, other external cues can be explored to guide the direction of organ growth. Providing a unilateral light source can guide the shoot growth, but prolonged root exposure to light causes a stress response, affecting growth and development, and also affecting the response to other environmental factors. Here, we have investigated how the protection of the root from light exposure, while the shoot is illuminated, influences the direction of root growth in microgravity. We report that the light avoidance mechanism existing in roots guides their growth towards diminishing light and helps establish the proper longitudinal seedling axis in simulated microgravity conditions. This process is regulated by flavonols, as shown in the flavonoid‐accumulating mutant transparent testa 3, which shows an increased correction of the root growth direction in microgravity, when the seedling is grown with the root protected from light. This finding may improve the efficiency of water and nutrient sourcing and photosynthesis under microgravity conditions, as they exist in space, contributing to better plant fitness and biomass production in space farming enterprises, necessary for space exploration by humans.

Summary: Seed dormancy governs germination timing, with both evolutionary and applied consequences. Despite extensive studies on the hormonal and genetic control of these processes, molecular mechanisms directly linking dormancy and germination remain poorly understood. By screening a collection of lines overexpressing Arabidopsis transcription factors, we identified ERF50 as a key gene to control dormancy and germination. To study its regulation, we measured seed‐related physiological parameters in loss‐of‐function mutants and carried out transactivation, protein interaction and ChIP‐PCR analyses. We found direct ERF50‐mediated repression of DOG1 and activation of EXPA2 transcription, which results in enhanced seed germination. Although ERF50 expression is increased by DOG1 in dormant seeds, ERF50 germination‐promoting activity is blocked by RGL2. The physiological, genetic and molecular evidence gathered here supports that ERF50 controls germination timing by regulating DOG1 levels to leverage its role as enhancer of seed germination, via RGL2 antagonism on EXPA2 expression. Our results highlight the central role of ERF50 as a feedback regulator to couple and fine‐tune seed dormancy and germination.

The yeast one‐hybrid system (Y1H) is used extensively to identify DNA–protein interactions. The generation of large collections of open reading frames (ORFs) to be used as prey in screenings is not a bottleneck nowadays and can be carried out in‐house or offered as a service by companies. However, the straightforward use of full gene promoters as baits to identify interacting proteins undermines the accuracy and sensitivity of the assay, especially in the case of multicellular eukaryotes. Therefore, it is paramount to implement procedures for efficient identification of suitable promoter fragments compatible with the Y1H assay. Here, we describe a workflow to identify biologically relevant conserved promoter fragments of Arabidopsis thaliana through simple and robust phylogenetic analyses. Additionally, we describe a manual method and its automated robotized version for rapid and efficient high‐throughput Y1H screenings of arrayed ORF libraries with the identified DNA fragments. Moreover, this method can be scaled up or down and used for yeast two‐hybrid screenings to search for possible interactors of proteins identified by the Y1H approach or any other protein of interest, altogether underscoring its suitability to build gene regulatory networks. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Selection of DNA baits for Y1H screeningsBasic Protocol 2: Y1H screenings with arrayed gene librariesAlternate Protocol: Automated screening with a liquid‐handling robot