DNA glycosylases initiate the base excision repair (BER) pathway by catalyzing the removal of damaged or mismatched bases from DNA. The Arabidopsis DNA glycosylase methyl-CpG-binding domain protein 4 like (MBD4L) is a nuclear enzyme triggering BER in response to the genotoxic agents 5-fluorouracil and 5-bromouracil. To date, the involvement of MBD4L in plant physiological processes has not been analyzed. To address this, we studied the enzyme functions in seeds. We found that imbibition induced the MBD4L gene expression by generating two alternative transcripts, MBD4L.3 and MBD4L.4. Gene activation was stronger in aged than in non-aged seeds. Seeds from mbd4l-1 mutants displayed germination failures when maintained under control or ageing conditions, while 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 seeds reversed these phenotypes. Seed nuclear DNA repair, assessed by comet assays, was exacerbated in an MBD4L-dependent manner at 24 h post-imbibition. Under this condition, the BER genes ARP, APE1L, and LIG1 showed higher expression in 35S:MBD4L.3/mbd4l-1 and 35S:MBD4L.4/mbd4l-1 than in mbd4l-1 seeds, suggesting that these components could coordinate with MBD4L to repair damaged DNA bases in seeds. Interestingly, the ATM, ATR, BRCA1, RAD51, and WEE1 genes associated with the DNA damage response (DDR) pathway were activated in mbd4l-1, but not in 35S:MBD4L.3/mbd4l-1 or 35S:MBD4L.4/mbd4l-1 seeds. These results indicate that MBD4L is a key enzyme of a BER cascade that operates during seed imbibition, whose deficiency would cause genomic damage detected by DDR, generating a delay or reduction in germination.

Seed vigor is an important trait for tobacco production. However, the evaluation of seed vigor using molecular biomarkers is scarcely reported in tobacco. In this study, the development of molecular marker isopropylmalate synthase NtIPMS was conducted to detect seed ageing degree and seed priming effect in tobacco. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression of NtIPMS was significantly induced at the initial imbibition stage during seed germination. The NtIPMS expression was positively correlated with the degree of seed ageing in non-pelleted and pelleted seeds. The mRNA level of NtIPMS was gradually increased with the increasing degree of seed ageing. The early best effect of gibberellin priming was observed in 30-h primed seeds, and the highest expression of NtIPMS was observed in 12-h primed seeds. The best stop time-point of seed priming is likely at the time 18 h after the relatively higher NtIPMS expression occurred during seed priming process. The NtIPMS mRNA detection has the potential usage as a potential molecular marker for the evaluation of seed vigor in tobacco.

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 lifespan or longevity of a seed is the time period over which it can remain viable. Seed longevity is a complex trait and varies greatly between species and even seed lots of the same species. Our scientific understanding of seed longevity has advanced from anecdotal \'Thumb Rules,\' to empirically based models, biophysical explanations for why those models sometimes work or fail, and to the profound realisation that seeds are the model of the underexplored realm of biology when water is so limited that the cytoplasm solidifies. The environmental variables of moisture and temperature are essential factors that define survival or death, as well as the timescale to measure lifespan. There is an increasing understanding of how these factors induce cytoplasmic solidification and affect glassy properties. Cytoplasmic solidification slows down, but does not stop, the chemical reactions involved in ageing. Continued degradation of proteins, lipids and nucleic acids damage cell constituents and reduce the seed\'s metabolic capacity, eventually impairing the ability to germinate. This review captures the evolution of knowledge on seed longevity over the past five decades in relation to seed ageing mechanisms, technology development, including tools to predict seed storage behaviour and non-invasive techniques for seed longevity assessment. It is concluded that seed storage biology is a complex science covering seed physiology, biophysics, biochemistry and multi-omic technologies, and simultaneous knowledge advancement in these areas is necessary to improve seed storage efficacy for crops and wild species biodiversity conservation.

Navua sedge (Cyperus aromaticus (Ridley) Mattf. & Kukenth) is a significant agricultural and environmental weed found in tropical island countries including north Queensland, Australia. It is a prolific seed producer and consequently forms a high-density seedbank, and therefore understanding the longevity and persistence of the seeds can provide critical information required for the management of this species. A laboratory-controlled artificial ageing experiment was conducted where the seeds were exposed to a temperature of 45 °C and 60% relative humidity for 125 days. Seeds were removed at various times (1, 2, 5, 9, 20, 30, 50, 75, 100 and 125 days) and their viability determined through standard germination tests. It took 20 days in the artificial ageing environment for the seeds to decline to 50% viability which indicates that Navua sedge has relatively short-lived persistent seeds. These findings will assist in developing a better understanding of the seedbank dynamics of this invasive species, allowing managers to tactically implement control strategies and prepare budgets for ongoing treatments, and have implications for the duration and success of management programs.

Seed ageing is associated with a high concentration of reactive oxygen species (ROS). Apple (Malus domestica Borkh.) seeds belong to the orthodox type. Due to a deep dormancy, they may be stored in dry condition at 5 °C for a long time, without viability loss. In the laboratory, artificial ageing of apple seeds is performed by imbibition in wet sand at warm temperature (33 °C). The aim of the work was to study nitric oxide (NO) as a seed vigour preservation agent. Embryos isolated from apple seeds subjected to accelerated ageing for 7, 14, 21 or 40 days were fumigated with NO. Embryo quality was estimated by TTC and MDA tests. ROS level was confirmed by NBT staining. We analysed the alteration in transcript levels of CAT, SOD and POX. NO fumigation of embryos of seeds aged for 21 days stimulated germination and increased ROS level which correlated to the elevated expression of RBOH. The increased total antioxidant capacity after NO fumigation was accompanied by the increased transcript levels of genes encoding enzymatic antioxidants, that could protect against ROS overaccumulation. Moreover, post-aged NO application diminished the nitro-oxidative modification of RNA, proving NO action as a remedy in oxidative remodelling after seeds ageing.

Fumarylacetoacetate hydrolase (FAH) proteins form a superfamily found in Archaea, Bacteria, and Eukaryota. However, few fumarylacetoacetate hydrolase domain (FAHD)-containing proteins have been studied in Metazoa and their role in plants remains elusive. Sequence alignments revealed high homology between two Arabidopsis thaliana FAHD-containing proteins and human FAHD1 (hFAHD1) implicated in mitochondrial dysfunction-associated senescence. Transcripts of the closest hFAHD1 orthologue in Arabidopsis (AtFAHD1a) peak during seed maturation drying, which influences seed longevity and dormancy. Here, a homology study was conducted to assess if AtFAHD1a contributes to seed longevity and vigour. We found that an A. thaliana T-DNA insertional line (Atfahd1a-1) had extended seed longevity and shallower thermo-dormancy. Compared to the wild type, metabolite profiling of dry Atfahd1a-1 seeds showed that the concentrations of several amino acids, some reducing monosaccharides, and δ-tocopherol dropped, whereas the concentrations of dehydroascorbate, its catabolic intermediate threonic acid, and ascorbate accumulated. Furthermore, the redox state of the glutathione disulphide/glutathione couple shifted towards a more reducing state in dry mature Atfahd1a-1 seeds, suggesting that AtFAHD1a affects antioxidant redox poise during seed development. In summary, AtFAHD1a appears to be involved in seed redox regulation and to affect seed quality traits such as seed thermo-dormancy and longevity.

Seed viability is an important trait in agriculture which directly influences seedling emergence and crop yield. However, even when stored under optimal conditions, all seeds will eventually lose their viability. Our primary aims were to describe factors influencing seed deterioration, determine the morphological, physiological, and biochemical changes that occur during the process of seed ageing, and explore the mechanisms involved in seed deterioration. High relative humidity and high temperature are two factors that accelerate seed deterioration. As seeds age, frequently observed changes include membrane damage and the destruction of organelle structure, an increase in the loss of seed leachate, decreases of respiratory rates and ATP production, and a loss of enzymatic activity. These phenomena could be inter-related and reflect the general breakdown in cellular organization. Many processes can result in seed ageing; it is likely that oxidative damage caused by free radicals and reactive oxygen species (ROS) is primarily responsible. ROS can have vital interactions with any macromolecule of biological interest that result in damage to various cellular components caused by protein damage, lipid peroxidation, chromosomal abnormalities, and DNA lesions. Further, ROS may also cause programmed cell death by inducing the opening of mitochondrial permeability transition pores and the release of cytochrome C. Some repairs can occur in the early stages of imbibition, but repair processes fail if sufficient damage has been caused to critical functional components. As a result, a given seed will lose its viability and eventually fail to germinate in a relatively short time period.

Seed aging is a complex biological process and its fundamentals and mechanisms have not yet been fully recognized. This is a key issue faced by research teams involved in the collection and storage of plant genetic resources in gene banks every day. Transcriptomic changes associated with seed aging in the dry state have barely been studied. The aim of the study was to develop an efficient protocol for construction of RNA-Seq libraries from long-term stored seeds with very low viability and low RNA integrity number (RIN). Here, barley seeds that have almost completely lost their viability as a result of long-term storage were used. As a control, fully viable seeds obtained in the course of field regeneration were used. The effectiveness of protocols dedicated to RNA samples with high and low RIN values was compared. The experiment concluded that library construction from low viable or long-term stored seeds with degraded RNA (RIN < 3) should be carried out with extraordinary attention due to the possibility of uneven degradation of different RNA fractions.

Seed longevity is a polygenic trait of relevance for agriculture and for understanding the effect of environment on the ageing of biological systems. In order to identify novel longevity genes, we have phenotyped the natural variation of 270 ecotypes of the model plant, Arabidopsis thaliana, for natural ageing and for three accelerated ageing methods. Genome-wide analysis, using publicly available single-nucleotide polymorphisms (SNPs) data sets, identified multiple genomic regions associated with variation in seed longevity. Reverse genetics of 20 candidate genes in Columbia ecotype resulted in seven genes positive for seed longevity (PSAD1, SSLEA, SSTPR, DHAR1, CYP86A8, MYB47 and SPCH) and five negative ones (RBOHD, RBOHE, RBOHF, KNAT7 and SEP3). In this uniform genetic background, natural and accelerated ageing methods provided similar results for seed-longevity in knock-out mutants. The NADPH oxidases (RBOHs), the dehydroascorbate reductase (DHAR1) and the photosystem I subunit (PSAD1) highlight the important role of oxidative stress on seed ageing. The cytochrome P-450 hydroxylase, CYP86A8, and the transcription factors, MYB47, KNAT7 and SEP3, support the protecting role of the seed coat during seed ageing.