https://orcid.org/0000-0003-3723-0398
Abstract
Plant senescence is a genetically controlled process that results in the programmed death of plant cells, organs or the entire plant. This process is essential for nutrient recycling and supports the production of plant offspring. Environmental stresses, such as drought and heat, can hasten senescence, reducing photosynthetic efficiency and significantly affecting crop quality and yield.
This invited review seeks to clarify the complex metabolic and biochemical transformations involved in plant senescence by explaining the mechanisms in a straightforward and connected manner. It focuses on key cellular processes, such as genetically programmed or stress-induced senescence, chlorophyll metabolism and nutrient recycling, while also exploring the roles of signalling molecules and pathways.
Understanding the complexities of plant senescence might help to manage crop ageing, address climate change and cut post-harvest losses. Enhancing crop resilience to stress and decelerating ageing can reduce the need for overproduction, thereby decreasing pollution and conserving resources. Tackling food waste, which constitutes about one-third of global supplies, is crucial for ensuring food security and fostering environmental sustainability.
INTRODUCTION
Senescence represents a vital and irreversible stage in plant growth. It involves a complex and highly organized sequence of events starting from the inception of the plant or its organs, leading up to their senescence and eventual death. Owing to the long lifetime of plants and trees, which makes studying their ageing and senescence challenging, researchers often focus on plant leaves to investigate this phenomenon. Leaf senescence is considered the final phase of organ development and represents a form of programmed cell death (PCD) (Miao and Zentgraf, 2007). This process, in fact, involves a shift from the assimilation of nutrients to their redistribution within the plant. Consequently, leaves shift from being nutrient-storing organs responsible for carbon fixation and nutrient absorption to nutrient-exporting organs, promoting the recycling of nutrients to actively growing plant tissues (Kim et al., 2016). The physiological processes throughout the life cycle of a plant are intricately linked. Initially, plants undergo a lag phase, characterized by a high but gradually decreasing growth rate as the plant establishes its root system and begins to develop leaves and stems. Next, plants experience rapid overall growth despite a continuous decline in the relative growth rate, significantly increasing in size and biomass. In the next stationary phase, their growth slows markedly, and the plants reach their maximum size and reproductive potential. During this phase, physiological activities also decelerate as the plants prepare for the subsequent decline stage, marked by a decrease in viability, indicating the onset of senescence. This phase is marked by substantial metabolic and physiological transformations, driven by genetic regulation and nutrient redistribution, which are essential for forming and supporting reproductive structures (Guiboileau et al., 2010; Thomas, 2013). Therefore, plant senescence usually encompasses physiological changes that facilitate growth, differentiation, adaptation, survival and reproduction. However, stress phenomena can also trigger senescence, causing premature ageing and metabolic shifts. This stress-induced senescence leads to the early degradation of chlorophyll and macromolecules, altering normal nutrient recycling and negatively impacting the overall fitness and productivity of the plant (Rapp et al., 2015). Both developmental and stress-induced senescence induce cellular mechanisms that regulate the metabolic pathways of DNA, amino acids, chlorophyll, sugars and reactive oxygen species (ROS) (Kanojia et al., 2021). Phytohormones and ROS act as internal signalling compounds able to mediate the responses of the plant to these stimuli (Mayta et al., 2019; Sasi et al., 2022). Developmental senescence in plants is designed to optimize the utilization of carbon, nitrogen and mineral nutrients to support growth and future generations. Through this process, nutrients are transferred efficiently from senescing tissues to growing and developing parts of the plant, which are crucial for the fitness and reproductive success of the plant. Conversely, premature senescence, which can be triggered by stress, functions as a survival mechanism to ensure the production of offspring in unfavourable conditions. However, this strategy often comes with a trade-off, leading to a decrease in both the quantity and quality of seeds, ultimately affecting the overall productivity and yield of plants (Zentgraf et al., 2022).
Although it is now known that leaf senescence significantly affects photosynthesis, nutrient mobilization, stress responses and productivity (Guo et al., 2021), many natural factors contributing to this process remain to be explored. These factors include the age of the organ, the interplay among various regulatory pathways, the dynamics of source–sink relationships, the remobilization of nutrients and the processes of anterograde and retrograde signal transduction (Li et al., 2023). Anterograde signalling involves transmission of information from the nucleus to organelles, which helps to regulate nuclear-encoded genes essential for organelle function. In contrast, retrograde signalling refers to the feedback from organelles, particularly chloroplasts and mitochondria, back to the nucleus. One important retrograde signal is hydrogen peroxide (H₂O₂), a molecule generated during stress or ageing processes. H₂O₂ acts as a signalling compound modulating gene expression in the nucleus, enabling the plant to cope with oxidative stress and properly regulate senescence (Foyer and Hanke, 2022; Zentgraf et al., 2022). Major progress has already been made in the transcriptomic study of leaf senescence, but analyses in the field of metabolomics remain relatively scarce, creating a gap in our comprehensive understanding of this complex process (Watanabe et al., 2013). Furthermore, a critical perspective connecting the roles of the two essential endosymbiotic cellular organelles, chloroplasts and mitochondria, to leaf senescence is lacking. Gaining insight into these physiological and metabolic changes is essential for managing the onset and timing of senescence in agricultural crops.
NEW INSIGHTS INTO EPIGENETIC CONTROL OF LEAF SENESCENCE
Leaf senescence represents the well-organized last phase in the development of leaves. Throughout this stage, chlorophylls, nucleic acids, lipids, proteins and other macromolecules are degraded systematically, and the nutrients released are reallocated to growing and storage organs. The developmental signals and environmental factors, such as abiotic stresses, which initiate the progression of plant senescence can cause significant genetic, physiological and metabolic changes, including the expression of senescence-associated genes (SAGs) (Guo and Gan, 2005). Genes that respond to stress, such as those encoding for Mitogen-Activated Protein Kinases (MAPKs) or transcription factors (TFs), such as WRKY and NAC, are central players during senescence progression (Zhang et al., 2021). WRKY is a family of plant TFs with a conserved WRKYGQK sequence, regulating plant development, responses to stress and senescence (Jiang et al., 2017). NAC, which stands for NAM (No Apical Meristem), ATAF1/2 (Arabidopsis Transcription Activation Factor 1/2), CUC2 (Cup-Shaped Cotyledon 2), is a large family of TFs playing a crucial role in different developmental processes and responses to stress (Shao et al., 2015). For instance, TFs such as ORE1 (NAC2) and ORE15 act as nodes in gene regulatory networks by activating other SAGs (Guo, 2013). Moreover, many SAGs encode hydrolytic enzymes and transporters that are responsible for breaking down and mobilizing various macromolecules.
Research in horticultural crops has highlighted that the initiation of SAGs transcription is intricately connected to histone modifications and DNA methylation patterns and is influenced by developmental signals and by both biotic and abiotic stress factors (Chachar et al., 2022). High-resolution epigenomic studies have revealed that the trimethylation of lysine 4 and acetylation of lysine 9 on histone H3 (H3K4me3 and H3K9ac) are markers of active transcription at the chromatin areas associated with SAGs (Yolcu et al., 2018). On the contrary, Histone deacetylase 15 (HDA15) and WHIRLY1 repress the transcription of SAGs, leaf senescence and flowering (Huang et al., 2022). WHIRLY1, also known as pTAC-1, is a protein able to ‘whirl’ around single-stranded DNA, protecting it and regulating stress-responsive genes, chloroplast DNA binding and stability, chloroplast nucleoid organization, and retrograde signalling (Krupinska et al., 2014; Lin et al., 2024). It has a significant role in maintaining the integrity and functionality of chloroplasts, essential for photosynthesis and overall plant health. In contrast, HDA9, which functions together with the SANT domain-containing protein POWERDRESS (PWR) and WRKY53, a TF that plays a key role in leaf ageing by triggering senescence when active, induces leaf senescence by reducing histone acetylation at specific gene loci, thus activating SAGs. The interaction between HDA9 and PWR is essential, because PWR supports the nuclear localization and functionality of HDA9, making this complex vital for managing leaf senescence (Chen et al., 2016). Long non-coding RNAs can also have crucial roles in regulating leaf senescence by engaging with different proteins and TFs to modulate the expression of SAGs. For example, MEKK1 can phosphorylate WRKY53, boosting its expression and promoting senescence. MAPKs, including MPK3 and MPK6, together with their upstream kinases MKK4 and MKK5, determine a regulatory cascade pivotal for control of senescence progression. However, the exact mechanisms modulating the age-dependent adjustments of these two H3 modifications at SAG locations remain largely unexplored, pointing to an area that warrants further investigation (Guo et al., 2021). Recently, also the possible role in the senescence of H3K27me3, a repressive histone mark that plays a significant role in modulating Arabidopsis gene expression, has emerged (Wang et al., 2019). Specifically, the H3K27me3 demethylase RELATIVE OF EARLY FLOWERING6 (REF6) has been identified as a key factor in senescence modulation. Without REF6, higher H3K27me3 levels are present, keeping SAGs inactive until the plant reaches a genetically programmed developmental stage, whereas, when present, REF6 can directly bind to the promoters of SAGs, such as NONYELLOWING1 (NYE1), increasing their expression, thus accelerating chlorophyll degradation and progression of the senescence process. This process ensures that the timing of senescence aligns with the lifecycle needs of the plant, allowing for effective photosynthesis and nutrient redistribution until it is necessary (Wang et al., 2019). Therefore, regulation by REF6 is crucial for the timely activation of senescence genes, which prevents premature ageing and optimizes resource allocation within the plant. Ding et al. (2022) demonstrated that SlJMJ4, a H3K27me3 demethylase found in tomatoes, when activated by exposure of plants to dark conditions and abscisic acid (ABA) stress, accelerates chlorophyll breakdown and senescence. Likewise, in Arabidopsis, H3K27me3 demethylases, such as JMJ30 and ELF6, are essential for the thermal stress response, highlighting the critical function of histone modifications in environmental adaptation and senescence (Yamaguchi, 2021). Recently, it has been suggested that H3K27me3 is a repressive chromatin mark also involved in regulating post-harvest senescence. Rogers and co-workers found that H3K27me3 levels are regulated dynamically during storage, influencing the activation of genes associated with stress responses, metabolism and cell wall modifications. These changes suggest the important function of epigenetic control in altering gene expression patterns during post-harvest in the development of plants and their responses to stress (Hilary Rogers, personal communication, unpublished data).
MOLECULAR INTERACTIONS AND OXIDATIVE STRESS IN CHLOROPLAST-MEDIATED SENESCENCE
Chlorophyll degradation and chloroplast signalling in leaf senescence
Chlorophyll degradation is indeed one of the earliest symptoms of leaf ageing. The precise regulation of chlorophyll levels, governed by the adaptative balance between chlorophyll biosynthesis and degradation, is vital for optimal photosynthesis and plant health (Wang et al., 2020).
Chlorophyll, a non-polar pigment, contains a porphyrin ring with a magnesium (Mg) atom at its centre and a long hydrophobic chain attached to one side. Chlorophyll biosynthesis occurs in the chloroplast via the C-5 pathway, starting with δ-aminolaevulinic acid from glutamic acid. The enzyme magnesium chelatase (E.C. 6.6.1.1) facilitates insertion of the Mg atom in the protoporphyrin ring. Chlorophyll a can be transformed to chlorophyll b and vice versa through the action of specific enzymes (Fig. 1). This interconversion, known as chlorophyll cycle, involves chlorophyllide a or b and hydroxymethyl chlorophyll a intermediates.
The interconversion between chlorophylls and the initial step of chlorophyll degradation. The enzymes are reported in yellow boxes along the pathways. The conversion of chlorophyll a or b into chlorophyllide a or b is catalysed by hydroxymethyl chlorophyll a reductase. In contrast, the conversion of chlorophyllide a or b into chlorophyll a or b is mediated by chlorophyll synthase. Chlorophyll b can be converted to hydroxylmethyl chlorophyll a by chlorophyllide a oxygenase, and geranylgeranyl diphosphate reductase can convert hydroxylmethyl chlorophyll a into chlorophyllide b or chlorophyll a into hydroxylmethyl chlorophyll a. Hydroxylmethyl chlorophyll a can be converted into chlorophyllide a by chlorophyll b reductase. Finally, chlorophyllide a can be converted into pheophytin by chlorophyllase.
Chlorophyll turnover is regulated by the enzyme chlorophyllase (chlase, chlorophyll-chlorophyllidohydrolase, E.C. 3.1.1.14), which hydrolyses chlorophyll by removing the phytol group and forming the pheophytin (Fig. 1) (Matile et al., 1999).
Chlase, which is situated in the inner portion of the chloroplast envelope, is permanently active throughout all phases of leaf development (Matile et al., 1997; Hörtensteiner et al., 2000). However, its activity increases significantly during senescence, particularly in the final stages, when chloroplasts lose their integrity (Fang et al., 1998). Chlase is believed to play a role in the initial step of chlorophyll catabolism (Fig. 1), but it also contributes to chlorophyll biosynthesis. During senescence, chlorophyll b degrades faster than chlorophyll a because it is converted into hydroxymethyl chlorophyll a, which is then transformed into chlorophyllide a by chlorophyll b reductase, and finally to chlorophyll a by chlorophyll synthase (E.C. 2.5.1.62).
The second step in chlorophyll breakdown involves transforming pheophytin to pheophorbide a through the action of pheophytinase (Fig. 2). Pheophorbide is the final green intermediate in chlorophyll breakdown. In subsequent steps, pheophorbide a oxygenase converts it into red chlorophyll catabolites (RCC), which are then reduced by RCC reductase to primary fluorescent chlorophyll catabolites (pFCC). These are processed further into non-fluorescent chlorophyll catabolites (NCCs), the final breakdown products, typically stored in the vacuoles (Hörtensteiner, 1999). The chlorophyll concentration in plants is influenced by many factors, including species, age, environment and plant hormones.
The main steps of chlorophyll degradation pathways and their localization. In the chloroplast, conversion of the pheophytin in pheophorbide a is catalysed by pheophytinase. Pheophorbide a is transformed by pheophorbide a oxygenase into red chlorophyll catabolites (RCC). These are transformed into primary fluorescent chlorophyll catabolites (pFCCs) by RCC reductase. In the cytoplasm, the pFCCs are transformed into modified or conjugated chlorophyll catabolites (FCCs) through demethylation and conjugations. These FCCs are then transferred into the vacuole and transformed into non-fluorescent chlorophyll catabolites (NCCs).
Research conducted on Arabidopsis has demonstrated that the paralogues of BALANCE of CHLOROPHYLL METABOLISM (BCM) produce functionally similar proteins that help to regulate the balance between chlorophyll production and its breakdown (Yamatani et al., 2022). In the initial stages of leaf development, BCM1 interacts with GENOMES UNCOUPLED 4 (GUN4) to boost the activity of Mg-chelatase, thereby enhancing chlorophyll production. Concurrently, binding of BCM1 with Mg-dechelatase facilitates the breakdown of this enzyme, preventing chlorophyll degradation.
As leaf senescence begins, the expression of BCM2 increases in comparison to BCM1, playing a crucial role in reducing chlorophyll degradation (Wang et al., 2020; Yamatani et al., 2022).
Therefore, post-translational regulation is vital for maintaining adequate chlorophyll levels to support the photosynthetic machinery by balancing its synthesis and degradation during leaf development (Wang et al., 2022). Besides, chlorophyll biosynthesis and stability are tightly coupled to photosynthetic assembling components. In particular, the proper folding of the D1 protein in photosystem II (PSII) happens only in the presence of chlorophyll a, which is, therefore, crucial for the functionality of photosystems (PSs) and the overall process of photosynthesis (Knoppová et al., 2022).
Chlorophyll a fluorescence as a tool for assessing senescence
As leaves transition from dark green to light green, the levels of chlorophyll decrease, along with a lesser reduction in various PSII subunits. Despite this, the FV/FM (variable fluorescence to maximum fluorescence) ratio, which indicates the maximum quantum efficiency of PSII, stays stable, indicating that the efficiency of photosynthesis remains unchanged even if the PSII complexes present in the leaves decrease as senescence progresses. The efficiency of PSII declines significantly only in the final senescence phase (Tamary et al., 2019).
The measurement of chlorophyll a fluorescence is frequently used in plant physiology to assess the level of stress in plants. It can also be used to monitor leaf senescence by examining the performance of photosynthetic systems. The fluorescence emitted by chlorophyll a from the leaves occurs within the electromagnetic radiation range of 680–740 nm and is generated by PSII. This energy release from the photosynthetic system is closely associated with the health and functionality of the leaves (Strasser et al., 1995). Chlorophyll molecules capture light energy, which can be used for photosynthesis, dissipated as heat or emitted as fluorescence. These three energy fates compete with each other, hence an increase in one of them results in a decrease in the others. Only ~1–2 % of the absorbed light is re-emitted as fluorescence. However, the chlorophyll a fluorescence induction curve decreases as leaf ageing advances. The elaboration of the intermediate data point of the curve allows the calculation of different indices obtained using the JIP test, that quantifies PSII functionality and provide diagnostic parameters regarding the photosynthetic efficiency of plants (Strasser and Strasser, 1995). The most critical photochemical reactions occur within leaves, and senescence influences vitality, functionality, chlorophyll a fluorescence and photosynthesis activities. Consequently, chlorophyll a fluorescence can be (Strasser et al., 1995) used to evaluate the senescence stage by assessing the vitality and functionality of leaves. Senescence can be induced by natural processes or external stresses, including harvesting or detaching vegetables and ornamentals. In leafy vegetables, senescence is a multifaceted physiological process involving cellular and biochemical changes, not only chlorophyll degradation. Although chlorophyll loss is a visible senescence marker, it is a late-stage event. This means that several crucial processes related to nutrient remobilization and metabolic decline have already occurred when chlorophyll loss becomes apparent. Instead, chlorophyll a fluorescence provides an earlier and more accurate assessment by detecting changes in PSII efficiency, mainly through the FV/FM ratio, which can detect photosynthetic decline before visible chlorophyll degradation. This allows for a more precise evaluation of senescence, especially in the early stages (Ferrante and Maggiore, 2007). In dark-adapted leaves, the JIP test parameters, derived from key points of the fluorescence induction curve, can be used to assess leaf senescence (Strasser et al., 1995). In leafy vegetables, leaf senescence is accurately described by the performance index (PI), dissipation energy (DIo/RC or DIo/CS) per cross-section or reaction centre, and density of active reaction centres across a given cross-sectional area (RC/CSm) at maximum fluorescence (FM). The intensity of the signals depends on the chlorophyll concentration in the tissue, which has been demonstrated by monitoring the leaf senescence of spinach (Spinacia oleracea L.) and lettuce (Lactuca sativa L.) stored at two different temperatures (e.g. 4 or 8 °C) (Baldassarre et al., 2011). The JIP test parameters were also used to probe senescence in cucumber (Cucumis sativus) cotyledons by studying alterations in PSII (Prakash et al., 2003). The senescence kinetics of field lettuce (Valerianella locusta) stored at 4 and 10 °C were analysed using chlorophyll a fluorescence parameters and JIP indices to evaluate quality and senescence. Various fluorescent indices, including PI, RC/CSm, DIo/CS, ETo/DIo and ABS/DIo, were used to highlight the differences and describe quality losses. Among these indices, PI showed a higher correlation with total chlorophyll and carotenoid concentrations. The senescence induction curve was lowered, and the I point, an intermediate step in electron transport whose absence signals photosynthetic disruption, was not identifiable, as observed by Ferrante and Maggiore (2007).
All measurable or calculable parameters obtained from chlorophyll a fluorescence are affected by senescence, and their responses vary in magnitude. The maximum quantum efficiency of PSII (FV/FM) declines slowly and is difficult to detect at the early stage of senescence, even in different plant species. Minimal fluorescence (Fo) is the fluorescence intensity at 20 µs, when the plastoquinone electron acceptor pool (Qa) is fully oxidized. An increase in Fo suggests damage to PSII, indicating an impaired ability to discharge all electrons effectively.
Net photosynthetic activity is the key physiological process in green organisms for supplying carbohydrates and energy. In plants, this process follows a daily pattern and changes during leaf development. Its maximum occurs when the leaf reaches its fully expanded stage and declines progressively during senescence. Older leaves are removed in horticultural crops such as tomatoes, because net photosynthesis declines while respiration continues to deplete energy sources for plant growth and productivity. Photosynthetic activity assesses leaf functionality by directly measuring CO2 assimilation in leaves. During senescence of stock (Matthiola incana L.) cut flowers, chlorophyll concentration, FV/FM ratio and net photosynthesis are closely related (Ferrante et al., 2012).
Role of Rubisco in nutrient remobilization during senescence
In leaves, ribulose-1,5-bisphosphate carboxylase (Rubisco) constitutes >50 % of the total soluble proteins, and during senescence, reduction in protein content leads to a decline in photosynthetic activity (Matile et al., 1999). Leaves have two components that can indicate both senescence and photosynthetic potential: proteins and chlorophyll. Studies suggest that high nitrogen fertilization can delay the decrease in net photosynthesis typically associated with senescence by extending the lifespan of plants and increasing leaf protein content (Chen et al., 2014).
Research has revealed that the expression levels of two Rubisco small subunit genes begin to decline ~5 days before the appearance of visible signs of leaf senescence (Breeze et al., 2011). This reduction in Rubisco gene expression aligns with the premature breakdown of stromal enzymatic proteins, such as Rubisco and GS2, which occurs before the breakdown of chlorophyll and thylakoid-associated proteins (Diaz-Mendoza et al., 2016). Although the genes involved in chlorophyll breakdown, including Chlorophyll b reductase (NYC1), Stay-Green1 (SGR1), Stay-Green2 (SGR2) and Pheophorbide a oxygenase (PaO), start to be expressed weeks before any visible signs of leaf tip yellowing appear, chlorophyll degradation happens only after the initiation of degradation of stromal proteins. This sequence of events emphasizes that nitrogen remobilization from stromal proteins is prioritized before the more extensive degradation of thylakoidal proteins and chlorophyll during the senescence of leaves (Diaz-Mendoza et al., 2016). Moreover, the disassembling of PSII complexes and photosynthetic apparatus follows a sequential process rather than occurring simultaneously (Jiao et al., 2020), enabling the remaining complexes to continue functioning effectively until the advanced stages of senescence. The D1 protein, a crucial constituent of PSII susceptible to light-induced damage, is continually damaged and repaired in mature leaves. Proteases such as Deg and FtsH rapidly degrade the damaged D1 protein, thereby reducing photo-oxidative pressure on PSII and ensuring that the photosystem resumes its functionality as soon as the D1 protein is resynthesized (Kato et al., 2012). As leaves age and undergo senescence, the turnover rate of D1 protein declines, leading to a reduced capacity for repair. This results in the dismantling of PSII and a consequent decline in the overall efficiency of photosynthetic mechanisms. This process is strongly influenced by changes in ABA levels, which regulate the onset and progression of leaf senescence. Besides, high ABA levels suppress the transcription of genes essential for D1 protein synthesis and PSII repair, such as OsFtsH2, psbA, psbB and psbC, leading to reduced D1 protein production and impaired repair, with lower D1 protein levels and less functional PSII in ageing leaves (Wang et al., 2016).
Alternative electron pathways in senescence
As the photosynthetic machinery is disassembled, alternative electron transfer pathways, such as those involving plastid terminal oxidase (PTOX), become increasingly significant. PTOX plays a crucial role in reducing oxygen to water, which helps to avoid electron transport chain (ETC) over-reduction and minimizes the production of ROS. This process is crucial for managing energy production and preventing oxidative damage during chloroplast degradation. The presence of PTOX in gerontoplasts (senescing chloroplasts) suggests its role in maintaining redox balance and ensuring controlled senescence. Furthermore, other electron transfer pathways, such as those mediated by the NAD(P)H dehydrogenase (NDH) complex, also help to manage electron flow during senescence. These pathways provide alternative routes for electron transport, thereby compensating for the loss of photosystem activity while aiding the transfer of nutrients from senescing leaves to other parts of the plant.
Alternative electron pathways, including cyclic electron flow around PSI and oxygen-dependent electron transport via PTOX, become increasingly significant to offset the reduced efficiency of the linear ETC. This reorganization helps to manage ROS production and maintain some level of ATP synthesis despite the overall decline in photosynthetic activity
ROS signalling and retrograde communication during senescence
ROS increase greatly when the disassembly of photosystems becomes prominent and their functional capacity strongly decreases. Among them, H2O2, in particular, has a crucial role in triggering and driving the progression of senescence, activating retrograde signals (from organelles to the nucleus), which trigger specific responses (Foyer and Hanke, 2022). Chloroplast-derived H2O2 initiates early senescence by affecting TFs and biosynthetic genes, whereas peroxisomal H2O2 is involved in signalling to the nucleus, altering oxidative stress-related gene expression (Zentgraf et al., 2022). This specificity in ROS signalling is achieved through the distinct reactivity of ROS with cellular components and the maintenance of different redox states within organelles. Overall, H2O2 acts as a key messenger in the regulatory networks that modulate plant senescence, enabling the transition from nutrient assimilation to redistribution, crucial for reproductive organ development and survival (Jajic et al., 2015). The redox chemistry of chloroplasts, including ROS propagation, modulates cell death and significantly impacts the timing and progression of senescence. During the shift from vegetative growth to reproductive development, plants transition from sequential senescence, in which nutrients are transferred from older to younger leaves, to monocarpic senescence, in which nutrients are redistributed from all rosette leaves to support the development of reproductive organs. Given that the retrograde signal exerted by H2O2 enables the transition from nutrient assimilation to the effective redistribution of nutrients, throughout the bolting and flowering period, intracellular H2O2 levels increase as plants transition to reproductive growth. These levels remain high in young and mature leaves, decreasing in older leaves once they no longer have nutrients to export (Zentgraf et al., 2022).
Veciana et al. (2022) showed that the protein genomes uncoupled 1 (GUN1) has a central role in retrograde signalling. It drives transcriptional changes triggered by these signals, ensuring that nuclear gene expression aligns with the chloroplast status. When chloroplasts are dysfunctional, GUN1 helps to initiate a response that downregulates the transcription of photosynthesis-associated nuclear genes (PhANGs), thus reducing the synthesis of chloroplast proteins. GOLDEN2-LIKE 1 (GLK1) is a TF essential for the development of chloroplasts and the activation of PhANGs, promoting the formation of chloroplasts and photosynthetic machinery (Zheng et al., 2024). BBX16, another transcription factor regulated by GLK1, is involved in cotyledon development and influences PhANGs expression. When chloroplast function is compromised, retrograde signalling involving GUN1 represses GLK1 expression to prevent further chloroplast development in stress conditions (Veciana et al., 2022).
Martín (2023) studied senescence-related retrograde signalling through experiments with Norflurazon, a herbicide that inhibits carotenoid biosynthesis, leading to photooxidative damage in chloroplasts and uncontrolled production of ROS. Their study showed that retrograde signals also influence post-transcriptional regulation through alternative splicing, which is a process that enables a single gene to produce multiple mRNA variants, a mechanism through which a single gene generates different protein isoforms. This process adjusts the mRNA transcripts produced, often leading to the degradation of those no longer needed via the nonsense-mediated decay pathway. This regulation ensures that the production of chloroplast proteins is tightly controlled. Light signals and retrograde signals converge to control chloroplast protein synthesis and senescence. Light positively regulates chloroplast development and function, often opposing the effects of retrograde signals. In particular, light and retrograde signals antagonistically regulate the splicing of mRNAs for chloroplast proteins. Although retrograde signals often lead to accumulation of unproductive mRNAs, light promotes production of functional mRNAs. This balance ensures that chloroplast biogenesis and function are appropriately modulated according to environmental conditions (Martín, 2023). Therefore, retrograde signals from chloroplasts and light signals converge to finely tune the expression of chloroplast proteins, ensuring proper plant growth and responsiveness to environmental changes. This regulation is essential for controlling the initiation and progression of senescence, emphasizing the intricate relationship between chloroplast activity and plant ageing. All these signals give the start to chloroplast degradation, during which not only pigments and proteins but also lipids break down through various pathways. The degradation of chlorophyll and galactolipid begins in the membranes of thylakoids, producing by-products such as phytol and fatty acids. To prevent toxicity, these are esterified within plastoglobules, with the hydroxyl group -OH-C20H40O from phytol reacting with the carboxyl group -COOH of the fatty acid palmitic acid, leading to the creation of an ester bond and the release of a water molecule, C15H31COO−C20H39 + H2O. These are then eliminated through self-digestion within the chloroplasts, in the vacuoles or by microautophagy. The chlorophyll without phytol, known as the first non-toxic product of chlorophyll degradation (pFCC), is transported from the stroma of the chloroplast to the cytosol. There, it is converted into a modified fluorescent chlorophyll catabolite (mFCC) before being transported to the vacuole, where it undergoes further breakdown into non-fluorescent chlorophyll catabolite (NCC) (Domínguez and Cejudo, 2021).
Chloroplast protein degradation occurs through three primary mechanisms in plants. The first process, autophagy, especially significant in conditions of carbon scarcity and during leaf senescence, involves the formation of autophagosomes. These structures encapsulate cytosolic components, including stromal chloroplast proteins, transporting them to the vacuole, where they are broken down (Wan and Ling, 2022). The second process, independent of the autophagy pathway, involves the formation of senescence-associated vacuoles. These are small acidic vacuoles characterized by high proteolytic activity, which contain proteases, such as SAG12, able to degrade specific stromal chloroplast proteins (e.g. Rubisco and GS), but not thylakoid proteins (e.g. D1 and LHCII protein components) (Otegui, 2018). Additionally, in Arabidopsis, a Chloroplast Vesiculation (CV) protein pathway plays a crucial role in chloroplast protein degradation in stress conditions. The CV protein triggers the formation of vesicles that contain both stromal and thylakoid membrane proteins, which are subsequently delivered to the vacuole to be degraded. This mechanism is also independent of autophagy and senescence-associated vacuoles. Overexpression of CV accelerates the degradation of chloroplasts and the senescence of leaves, whereas silencing CV enhances stress tolerance and delays chloroplast turnover (Wang and Blumwald, 2014). Chloroplasts might also play a role in cell death by releasing cytochrome f, similar to the function of cytochrome c in animal cells. Cytochrome f in rice leaves interacts with proteasome components, inducing caspase-like activity and cell death (Wang and Blumwald, 2014).
ROLE OF CHLOROPLAST AND MITOCHONDRIAL OXIDATIVE STRESS IN SENESCENCE
During senescence, reducing antioxidants accelerates the degradation process by increasing ROS levels. For example, the decrease in tocopherol levels and the deactivation of thylakoid-bound ascorbate peroxidase (tAPX) can lead to photooxidative stress and accumulation of ROS, which modulate SAGs and promote early senescence and breakdown of chloroplasts and other cellular components (Juvany et al., 2013). On the contrary, in stay-green plants, senescence onset is delayed or progresses more slowly owing to the regulated breakdown of chlorophyll, which keeps leaves green for longer and maintains photosynthetic activity. This delay is managed by stay-green (SGR) genes and is influenced by ROS signalling (Thomas and Ougham, 2014).
Stay-green phenotypes and hormonal regulation
Stay-green plants typically exhibit delayed ageing and senescence and cope better with environmental stresses, such as salinity, drought or heat, resulting in increased yields and more robust crops. Cosmetic stay-green plants exhibit an anomaly in the initial stages of chlorophyll breakdown. In contrast, functional stay-green plants experience a delay in transitioning from the canopy phase of carbon storage to nitrogen mobilization. As a result, traits of ageing and senescence appear more gradually (Thomas and Ougham, 2014).
In functional ones, the typical stay-green phenotype is attributable to alterations in hormone metabolism, such as that of cytokinins (CK) and ethylene (ET), and signalling pathways and/or involves mutations in the WRKY and NAC TF families. Accordingly, Sekhon et al. (2019) identified key genes involved in the stay-green trait, including an NAC TF, trehalose-6-phosphate synthase, and other enzymes involved in cell wall formation. These genes maintain the stay-green phenotype by supporting source–sink communication. Additionally, studies on Cys protease mutants in Arabidopsis suggest similar mechanisms in maize for prolonging the green state during senescence (Sekhon et al., 2019). Likewise, in rice, Shin et al. (2020) demonstrated that variations in the regulatory promoter region of the SGR gene (OsSGR) influenced hormonal signalling and chlorophyll degradation, delaying senescence and improving yield. In indica subspecies, higher OsSGR expression accelerates senescence, whereas japonica ones show delayed senescence, allowing for prolonged photosynthesis. Thus, introducing japonica alleles into indica subspecies pathway improved grain yield owing to extended photosynthetic activity (Shin et al., 2020). Enhancing functional stay-green traits through selective breeding or genetic modifications, such as introducing specific alleles, can boost crop yields by extending photosynthetic activity. This approach can lead to more sustainable agricultural practices when combined with strategies that improve sink capacity, environmental resilience and effective crop management. Moreover, targeted CRISPR-Cas-based gene-editing techniques, which are increasingly accepted, offer a precise way to achieve these traits with minimal impact on public perception (Chen et al., 2024). Furthermore, given that climate change influences the timing and progression of senescence, understanding stay-green traits is crucial for developing crops that can adapt to future climatic changes (Thomas and Ougham, 2014; Pixley et al., 2023).
Antioxidant defence mechanisms during senescence
Notably, even as plant tissues undergo senescence or die, they continue to synthesize antioxidants, including those derived from degraded chlorophyll during post-harvest, such as α-tocopherol in Brassica rapa L. subsp. sylvestris (Annunziata et al., 2012). This conversion involves chlorophyllase enzymes and pheophytin pheophorbide hydrolase (PPH), which remove the phytol chain from chlorophyll, which is then converted into phytyl diphosphate by the enzymes VITAMIN E DEFICIENT5 and 6 (VTE5 and VTE6). Phytyl diphosphate is a crucial precursor for tocopherol biosynthesis, which helps to maintain the antioxidant defences of the plant during senescence (Vom Dorp et al., 2015). This biosynthesis is a key part of the mechanism to manage oxidative stress and maintain cellular function by recycling degradation products to bolster antioxidant activity, even during the last stage of leaf senescence.
Also, γ-aminobutyric acid (GABA) can have a crucial role in postponing plant senescence, not only by boosting antioxidant enzyme activity (Khan et al., 2021) but also by acting as a potent antioxidant by scavenging ROS, such as H2O2, singlet oxygen and superoxide anion radicals. This superior scavenging activity helps to stabilize and protect thylakoids and other macromolecules from oxidation (Carillo, 2018). Moreover, the synthesis of GABA from glutamate by GABA decarboxylase consumes protons, thus helping to stabilize cytosolic pH during stress conditions and preventing cellular damage. Additionally, the GABA converted into succinic acid by GABA shunt feeds the Krebs cycle, replenishing its intermediates and playing an anaplerotic role essential for maintaining metabolic homeostasis and energy production, especially in the last phases of progression of senescence (Carillo, 2018; Khan et al., 2021).
Kanojia et al. (2021), combining systems biology and molecular genetics studies, highlighted that metabolites such as chlorophylls and sugars, along with amino acids, stress tolerance metabolites and hormones, such as gibberellins and cytokinins, are abundant in young leaves, but decrease as leaves mature and age. In contrast, in ageing leaf cells, oxidative stress and hormones involved in the stress response, such as ET, ABA, salicylic acid (SA) and jasmonic acid (JA), increase greatly. Premature senescence onset is associated with elevated levels of ROS, which increase the leaf cell sensitivity to oxidative stress with a concurrent reduction in the activity of enzymes involved in ROS scavenging. Accordingly, during senescence, the expression of genes responsible for ET production is enhanced (Ghimire et al., 2023). Jing et al. (2002) introduced the idea of the ‘senescence window’, which explains leaf ageing through three developmental phases. In the first phase of development, leaves function as sink tissues that absorb and store nutrients, focusing on growth, contrasting premature ageing even in the presence of ET. As leaves mature, they become increasingly responsive to senescence-inducing factors, making them more competent to initiate ageing. In the final phase, leaves accumulate age-related cues that trigger senescence even in optimal conditions. However, leaves can still respond to signals that delay or reverse ageing. The discovery of molecular markers linked to leaf senescence was essential, enabling a comprehensive analysis at the transcriptional level. The role of ethylene in age-dependent leaf senescence was first demonstrated using two genes essential in the process of leaf senescence, SAG2 and SAG12. SAG2 is activated early in the senescence process and signals the onset of ageing in leaves; in contrast, SAG12 acts as a late-stage marker, encoding a cysteine protease that helps to break down cellular components, allowing the plant to recycle nutrients from old leaves to support new growth and seed development (Guo, 2013). As mentioned earlier, the initiation of leaf senescence is characterized by a decrease in hormones that maintain greenness, accompanied by a sequential increase in levels of ET, ABA, JA and, finally, SA. The levels of hormones and the cell responsiveness to them vary throughout development, depending on the specific stage of ageing (Guiboileau et al., 2010). This coordination provides the plant with phenotypic plasticity, enabling adaptation to developmental cues and/or adverse environmental conditions, allowing, only when necessary, dismantling of leaf cells to remobilize nutrients and guarantee the success of reproduction (Schippers et al., 2015).
Interplay of ethylene, salicylic acid and nitric oxide in senescence
Although it is well established that ET and SA are key regulators in facilitating the process of senescence, the precise nature of their interaction remains unclear. Research has demonstrated that the transcription factor ETHYLENE INSENSITIVE3 (EIN3), together with its homologue ETHYLENE INSENSITIVE3-LIKE 1 (EIL1), both crucial in ET signalling, promote senescence by triggering the activation of genes responsible for chlorophyll breakdown and the master senescence regulator ORE1/NAC2 (Li et al., 2013). These factors are also necessary for leaf senescence induced by SA in Arabidopsis (Li et al., 2013; Dolgikh et al., 2019). The name ORE1 is derived from the Korean word ‘oresara’, meaning ‘long-lived’, because the TF was initially associated with longevity rather than senescence (Woo et al., 2004). Moreover, ET has been found to amplify the SA senescence-promoting effects. WRKYs and TGACG motif-binding factors (TGAs) interact with the master regulator of SA signalling Nonexpressor of Pathogenesis-Related genes 1 (NPR1) to modulate the (immune) stress response and senescence (Mishra et al., 2024). Biochemical studies indicate that NPR1 also engages with EIN3 to enhance its ability to activate transcription. This suggests a cooperative function of SA and ET in promoting senescence (and the stress response), unlike their antagonistic interactions observed in other biological contexts (Wang et al., 2021; Yu et al., 2021).
ET has a crucial role in the modulation of fruit ripening and senescence. Production of ET shifts from an initial self-inhibitory phase [System 1 (S1)] to a self-amplifying phase [System 2 (S2)] in climacteric fruits. During S1, ET levels remain low, supporting basic growth and development. In contrast, S2 involves a dramatic self-catalytic increase in ET production, causing a burst in respiration known as the climacteric. This process is similar to the senescence window concept, in which ET exerts its effects when cells acquire responsiveness to its signals (Ferrante and Francini, 2006). This transition, involving complex genetic, hormonal and transcriptional regulatory mechanisms, ensures synchronized maturation and the development of ripening markers, including changes in colour, texture, sweetness and flavour (Hewitt and Dhingra, 2020).
SA is instrumental in the respiratory climacteric linked to the ET burst. In fact, as ET production peaks, SA boosts the synthesis and functionality of alternative oxidase (AOX) in the mitochondrial ETC, helping to manage the enhanced electron flow and ROS generation to promote a balanced ripening (Hewitt and Dhingra, 2020). AOX transfers electrons from ubiquinol to oxygen when ubiquinol is over-reduced, thus bypassing complex III or IV and not directly contributing to increasing the electron gradient formation for ATP synthesis. In this way, AOX dissipates excess reducing power, particularly in stress conditions and/or senescence, thus preventing the accumulation of reduced ubiquinone (Vanlerberghe, 2013). Therefore, the SA modulation of AOX might help to manage the increased oxidative stress associated with dismantling cellular components and nutrient recycling, preventing the overproduction of ROS, maintaining the redox balance, and supporting metabolic flexibility during senescence progression. However, when endogenous SA starts to accumulate, it enhances the production of mitochondrial ROS and nitric oxide (NO) and that of free calcium levels in the cytoplasm, thus inducing the plasma membrane NADPH oxidase complex to generate ROS. In conjunction with NO, this can enhance senescence and cell death or independently activate defence mechanisms. Additionally, SA induces mitochondrial complex III to produce superoxide (O2•−). This superoxide can react with NO to form peroxynitrite (ONOO−), affecting the redox status of the cell and potentially triggering cell death. The O2•− can also be converted to H2O2 by superoxide dismutase. When ROS increase to critical levels, SA might stimulate AOX activity to prevent oxidative damage, thus reducing mitochondrial ROS levels, maintaining redox balance and preventing cell death (Poór, 2020; Hussain et al., 2022). This dual role of SA ensures that it protects cells from oxidative damage while supporting controlled ROS-dependent signalling mechanisms. However, at high concentrations of SA, ET might amplify the oxidative stress response initiated by SA, increasing H2O2 production and contributing significantly to PCD and senescence pathways. SA further induces oxidative stress, particularly affecting mitochondria, and generating ROS in a concentration-dependent manner. A two-phase ROS burst model describes the process, whereby an initial ROS peak, possibly from NADPH oxidase or mitochondria, activates mitochondrial permeability transition pores (PTP), thus causing a second larger ROS burst involving chloroplasts. SA also promotes PTP opening, causing mitochondrial swelling, inhibition of the mitochondrial ETC, increased ROS and NO production, lipid peroxidation and membrane potential (ΔΨ) collapse, leading to cytochrome c release and PCD. Mitochondrial hexokinases (HXKs) are crucial regulators of the PTP in response to SA (Poór, 2020). Ca2+-binding proteins and calcium-dependent protein kinases (CDPKs) can also participate in the modulation of ROS levels, accelerating PCD in senescing tissues (Yang et al., 2022).
Recent research underscores the dual role of NO in the senescence process: it can either delay ageing by neutralizing ROS or accelerate their production by triggering oxidative stress (Guo et al., 2023). NO can modulate phytohormones, such as ET, ABA or JA, integrating itself into the hormonal regulation of ageing. NO breaks seed dormancy by stimulating ET production, influencing fruit ripening by altering the nitro-oxidative environment and regulating ET pathway genes. This dual role in promoting and delaying senescence highlights the critical importance of NO in plant development and stress adaptation (Guo et al., 2021). Fig. 3 illustrates the progression of senescence in plants, driven by both abiotic stresses and developmental cues. It shows how endogenous factors, such as ROS, ET and NO, contribute to the onset of senescence, leading to chlorophyll breakdown, nutrient remobilization and, ultimately, cell death.
Progression from healthy plants to senescing plants driven by abiotic stresses (e.g. drought, salinity, temperature changes) and developmental cues (e.g. ageing, reproductive phase). These factors initiate oxidative stress and hormonal imbalances involving ethylene (ET), salicylic acid (SA) and nitric oxide (NO), which, in turn, activate retrograde signalling (through ROS, GUN1 and ABA). This signalling cascade triggers cellular damage and marks the onset of senescence. During this phase, the plant undergoes chlorophyll degradation, nutrient remobilization and the activation of senescence-associated genes (SAGs). These processes lead to the dismantling of chloroplasts and protein degradation. The final stage of this progression is programmed cell death (PCD), regulated by the coordinated actions of hormones such as ABA, ET, SA and NO, controlling the timing and advancement of these physiological changes.
METABOLITE DYNAMICS IN SENESCING LEAVES
Mitochondria are essential for energy production and contribute significantly to the catabolism and redistribution of nutrients, especially N-rich metabolites. Intermediates from glycolysis and the Krebs cycle are essential for synthesis of amino acids, thus connecting central metabolic pathways with the production of proteins and other crucial cellular activities.
During leaf senescence, plants undergo significant metabolic changes that are crucial for nutrient redistribution. In a gas chromatography–mass spectrometry study, Chrobok et al. (2016) analysed the changes in metabolism associated with developmental leaf senescence. They highlighted that as senescence progresses, there is a complex metabolic reprogramming involving sugars, organic acids and amino acids. In particular, Ala, Asn, Glu, Gly, Pro and Ser decreased in abundance, although aromatic and branched-chain amino acids remained high. Branched-chain amino acids under stress can function as compatible solutes and contribute as alternative electron donors for the mitochondrial ETC, thus preserving mitochondrial functionality (Woodrow et al., 2017). Both Chrobok et al. (2016) and Launay et al. (2019) found that the number of mitochondria in Arabidopsis decreases by ~30 %, but integrity is conserved until the last stage of senescence. The catabolism of amino acids and lipids redirects intermediates back to glycolysis and the tricarboxylic acid cycle; in this way, mitochondria sustain primary metabolic functions until late phases of developmental leaf senescence and host essential catabolic processes for nutrient reallocation (Li et al., 2017). This process underscores the amphibolic nature of cellular respiration, whereby metabolic pathways serve dual roles in the breakdown (catabolism) and synthesis (anabolism) of molecules, facilitating a dynamic balance between energy production and biosynthesis within the cell even during the progression of senescence. Accordingly, Watanabe et al. (2013) found that leaf tips turn yellow/senescent first, but protect themselves while exporting nutrients. In fact, they accumulate Pro, Ala and GABA in leaf tips as stress-induced osmoprotectants, while synthesizing amides crucial for C and N transport.
From the metabolic studies of tobacco leaves in different positions (e.g. upper, middle and lower), Li et al. (2017) showed that light can positively regulate the chloroplast, preserving its functions and contrasting with the opposing effects of retrograde signals/senescence. Combining transcriptomic and metabolomic analyses in Arabidopsis, Law et al. (2018) tried to unravel the metabolic adaptations in response to various darkening treatments. They proved that leaves exposed to localized darkening trigger senescence far more quickly than leaves from an entirely darkened plant. In particular, individually darkened leaves enter senescence significantly faster than leaves from a fully darkened plant. Specifically, individually darkened leaves undergo rapid senescence characterized by heightened metabolic activity and significant transport of amino acids and sugars from the darkened leaf to other parts of the plant. Mitochondrial activity remains elevated, facilitating nutrient retrieval and supporting the degradation of the leaf, providing nutrients to other parts of the plant. In a plant in complete darkness, the sensing of carbon starvation induces metabolic shifts, resulting in the build-up of amino acids with a high nitrogen-to-carbon ratio. This mechanism facilitates the storage of cytotoxic ammonium and preserves a nitrogen pool. The leaves of a plant in complete darkness enter a suppressed metabolic state, conserving photosynthetic capacity and reducing respiration rates to survive prolonged darkness. Thus, the model of Law et al. (2018) identifies two distinct responses to darkening in plants: individually darkened leaves initiate rapid senescence characterized by increased metabolic activity and nutrient transport, whereas complete darkness triggers a survival strategy through metabolic suppression, conserving resources and sustaining essential functions until light returns.
CONCLUSION
Recent research into the mechanisms of leaf senescence has unravelled a complex interplay between catabolic and anabolic processes. During senescence, the degradation of chlorophyll, carbohydrates, proteins, lipids and nucleic acids facilitates the redistribution of resources to developing plant parts. This process is regulated by various molecules, including phytohormones and ROS, which exert retrograde signalling for starting senescence but also help to manage the oxidative stress associated with cellular breakdown. Accordingly, studies have emphasized the dual function of NO in senescence, which can either delay ageing by scavenging ROS or promote senescence by inducing oxidative stress. Additionally, the modulation of AOX by SA is crucial in maintaining redox balance and preventing excessive ROS accumulation during senescence. Recent research shows that leaf tips, plant lower leaves and leaves darkened individually undergo rapid senescence characterized by high metabolic activity and nutrient transport. In contrast, whole plant darkening triggers metabolic suppression, conserving resources and reducing respiration to endure prolonged darkness. These findings highlight the adaptive strategies of plants to manage senescence in varying developmental and environmental conditions. Understanding these intricate processes and regulatory networks is vital for developing strategies to boost crop yield, improve stress resilience and ensure sustainable agricultural practices. Future research integrating transcriptomic, metabolomic and epigenetic data will be essential to understand senescence fully and to develop targeted interventions for crop improvement.
