Protein degradation and nitrogen remobilization during leaf senescence

Leaf senescence, a type of programmed cell death, is a complex and highly regulated process that involves the degradation of macromolecules, including proteins, nucleic acids, and lipids. Nutrients, especially nitrogen, are re-mobilized from senescing leaves to newly developing tissues or reserve organs. Our review focuses on three pathways for protein breakdown and the resorption of N during this process: the ubiquitin/proteosome system, the chloroplast degradation pathway, and the vacuolar and autophagic pathway. We propose that two relative biochemical cycles exist for amino acid recycling and N-export — the GS/GOGAT cycle and the PPDK-GS/GOGAT cycle.     

Leaf senescence and abiotic stresses share reactive oxygen species-mediated chloroplast degradation

Leaf senescence is a genetically programmed decline in various cellular processes including photosynthesis and involves the hydrolysis of macromolecules such as proteins, lipids, etc. It is governed by the developmental age and is induced or enhanced by environmental stresses such as drought, heat, salinity and others. Internal factors such as reproductive structures also influence the rate of leaf senescence. Reactive oxygen species (ROS) generation is one of the earliest responses of plant cells under abiotic stresses and senescence. Chloroplasts are the main targets of ROS-linked damage during various environmental stresses and natural senescence as ROS detoxification systems decline with age. Plants adapt to environmental stresses through the process of acclimation, which involves less ROS production coupled with an efficient antioxidant defence. Chloroplasts are a major site of protein degradation, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is rapidly and selectively degraded during senescence and stress. The process of protein degradation is initiated by ROS and involves the action of proteolytic enzymes such as cysteine and serine proteases. The mechanism of Rubisco degradation still remains to be elucidated. The molecular understanding of leaf senescence was achieved through the characterization of senescence-associated genes and various senescence mutants of Arabidopsis, which is a suitable model plant showing monocarpic senescence. The regulation of senescence involves many regulatory elements composed of positive and negative elements to fine-tune the initiation and progression of senescence. This review gives an overview on chloroplast protein degradation during leaf senescence and abiotic stresses and also highlights the role of ROS management in both processes.     

Signal transduction in leaf senescence

Leaf senescence is a complex developmental phase that involves both degenerative and nutrient recycling processes. It is characterized by loss of chlorophyll and the degradation of proteins, nucleic acids, lipids, and nutrient remobilization. The onset and progression of leaf senescence are controlled by an array of environmental cues (such as drought, darkness, extreme temperatures, and pathogen attack) and endogenous factors (including age, ethylene, jasmonic acid, salicylic acid, abscisic acid, and cytokinin). This review discusses the major breakthroughs in signal transduction during the onset of leaf senescence, in dark- and drought-mediated leaf senescence, and in various hormones regulating leaf senescence achieved in the past several years. Various signals show different mechanisms of controlling leaf senescence, and cross-talks between different signaling pathways make it more complex. Key senescence regulatory networks still need to be elucidated, including cross-talks and the interaction mechanisms of various environmental signals and internal factors.     

Does autophagy mediate age-dependent effect of dietary restriction responses in the filamentous fungus Podospora anserina?

Autophagy is a well-conserved catabolic process, involving the degradation of a cell''s own components through the lysosomal/vacuolar machinery. Autophagy is typically induced by nutrient starvation and has a role in nutrient recycling, cellular differentiation, degradation and programmed cell death. Another common response in eukaryotes is the extension of lifespan through dietary restriction (DR). We studied a link between DR and autophagy in the filamentous fungus Podospora anserina, a multicellular model organism for ageing studies and mitochondrial deterioration. While both carbon and nitrogen restriction extends lifespan in P. anserina, the size of the effect varied with the amount and type of restricted nutrient. Natural genetic variation for the DR response exists. Whereas a switch to carbon restriction up to halfway through the lifetime resulted in extreme lifespan extension for wild-type P. anserina, all autophagy-deficient strains had a shorter time window in which ageing could be delayed by DR. Under nitrogen limitation, only PaAtg1 and PaAtg8 mediate the effect of lifespan extension; the other autophagy-deficient mutants PaPspA and PaUth1 had a similar response as wild-type. Our results thus show that the ageing process impinges on the DR response and that this at least in part involves the genetic regulation of autophagy.     

Senescence of Unfertilised Flowers in Epiphyllum Hybrids

Epiphyllum hybrids served as a model for the study of reserve remobilisation from unfertilised flowers to the mother stem tissues. Early phases of the tube senescence characterised by wilting were connected with degradation and transfer of reserve substances to the somatic organs of the mother plant. The degradation process began in perianth and stamens and continued through the successive zones (receptacular, pericarpellar and pedicellar) of the flower tube. The phloem-mediated backward substance transport was naturally indicated by the red pigment of the perianth -- cactorubin, while integrity of cells and tissues and green colour of the flower tube were still preserved. For the later phases of senescence the loss of permeability and successive breaking of the cell integrity, connected with the colour change of the tube from green to red was evident. The functioning of vascular bundles especially their phloem parts conducting dissolved substances to the sinks in mother stem organs were preserved until late stages of senescence. The recycling and remobilisation of nutrients from all parts of unfertilised ovary and ovules may be considered as a part of the life strategy in the family Cactaceae as well as in other taxa evolutionarily adapted to life in extreme environmental conditions.     

Correlation of leaf senescence and gene expression/activities of chlorophyll degradation enzymes in harvested Chinese flowering cabbage (Brassica rapa var. parachinensis)

Chinese flowering cabbage is one of the main leafy vegetables produced in China. They have a rapid leaf yellowing due to chlorophyll degradation after harvest that limits their marketing. In the present study, leaf senescence of the cabbages was manipulated by ethylene and 6-benzyl aminopurine (6-BA) treatment to investigate the correlation of leaf senescence and chlorophyll degradation related to gene expression/activities in the darkness. The patterns of several senescence associated markers, including a typical marker, the expression of senescence-associated gene SAG₁₂, demonstrated that ethylene accelerated leaf senescence of the cabbages, while 6-BA retarded this progress. Similar to the trends of BrSAG₁₂ gene expression, strong activation in the expression of three chlorophyll degradation related genes, pheophytinase (BrPPH), pheophorbide a oxygenase (BrPAO) and red chlorophyll catabolite reductase (BrRCCR), was detected in ethylene treated and control leaves during the incubation, while no evident increase was recorded in 6-BA treated leaves. The overall dynamics of Mg-dechelatase activities in all treatments displayed increasing trends during the senescence process, and a delayed increase in the activities was observed for 6-BA treated leaves. However, chlorophyllase activity as well as the expression of BrChlase1 and BrChlase2 decreased with the incubation in all treatments. Taken together, the expression of BrPPH, BrPAO and BrRCCR, and the activity of Mg-dechelatase was closely associated with the chlorophyll degradation during the leaf senescence process in harvested Chinese flowering cabbages under dark conditions.     

Autophagy is a key factor in maintaining the regenerative capacity of muscle stem cells by promoting quiescence and preventing senescence

Autophagy, a highly regulated cellular degradation and recycling process, can occur constitutively at a basal level, and plays an essential role in many aspects of cell physiology. A recently published study (see the related punctum in Autophagy, Vol. 12, No. 4) suggests that basal autophagy is also important for maintaining the regenerative capacity of muscle stem cells, and that the decline of autophagy with aging is the cause of entry into senescence from quiescence in satellite cells.     

The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana

The vacuole/lysosome serves an important recycling function during starvation and senescence in eukaryotes via a process called autophagy. Here bulk cytosolic constituents and organelles become sequestered in specialized autophagic vesicles, which then deliver their cargo to the vacuole for degradation. In yeasts, genetic screens have identified two novel post-translational modification pathways remarkably similar to ubiquitination that are required for autophagy. From searches of the Arabidopsis genome, we have identified gene families encoding proteins related to both the APG8 and -12 polypeptide tags and orthologs for all components required for their attachment. A single APG7 gene encodes the ATP-dependent activating enzyme that initiates both conjugation pathways. Phenotypic analysis of an APG7 disruption indicates that it is not essential for normal growth and development in Arabidopsis. However, the apg7-1 mutant is hypersensitive to nutrient limiting conditions and displays premature leaf senescence. mRNAs for both APG7 and APG8 preferentially accumulate as leaves senesce, suggesting that both conjugation pathways are up-regulated during the senescence syndrome. These findings show that the APG8/12 conjugation pathways have been conserved in plants and may have important roles in autophagic recycling, especially during situations that require substantial nitrogen and carbon mobilization.     

The roles of autophagy in development and stress responses in Arabidopsis thaliana

Autophagy is a dynamic process that involves the recycling process of the degradation of intracellular materials. Over the past decade, our molecular and physiological understanding of plant autophagy has greatly been increased. Most essential autophagic machineries are conserved from yeast to plants. The roles that autophagy-related genes (ATGs) family play in the lifecycle of the Arabidopsis are proved to be similar to that in mammal. Autophagy is activated during certain stages of development, senescence or in response to starvation, or environmental stress in Arabidopsis. In the progression of autophagy, ATGs act as central signaling regulators and could develop sophisticated mechanisms to survive when plants are suffering unfavorable environments. It will facilitate further understanding of the molecular mechanisms of autophagy in plant. In this review, we will discuss recent advances in our understanding of autophagy in Arabidopsis, areas of controversy, and highlight potential future directions in autophagy research.     

Plant organ senescence – regulation by manifold pathways

Senescence is the final stage of plant ontogeny before death. Senescence may occur naturally because of age or may be induced by various endogenous and exogenous factors. Despite its destructive character, senescence is a precisely controlled process that follows a well‐defined order. It is often inseparable from programmed cell death (PCD), and a correlation between these processes has been confirmed during the senescence of leaves and petals. Despite suggestions that senescence and PCD are two separate processes, with PCD occurring after senescence, cell death responsible for senescence is accompanied by numerous changes at the cytological, physiological and molecular levels, similar to other types of PCD. Independent of the plant organ analysed, these changes are focused on initiating the processes of cellular structural degradation via fluctuations in phytohormone levels and the activation of specific genes. Cellular structural degradation is genetically programmed and dependent on autophagy. Phytohormones/plant regulators are heavily involved in regulating the senescence of plant organs and can either promote [ethylene, abscisic acid (ABA), jasmonic acid (JA), and polyamines (PAs)] or inhibit [cytokinins (CKs)] this process. Auxins and carbohydrates have been assigned a dual role in the regulation of senescence, and can both inhibit and stimulate the senescence process. In this review, we introduce the basic pathways that regulate senescence in plants and identify mechanisms involved in controlling senescence in ephemeral plant organs. Moreover, we demonstrate a universal nature of this process in different plant organs; despite this process occurring in organs that have completely different functions, it is very similar. Progress in this area is providing opportunities to revisit how, when and which way senescence is coordinated or decoupled by plant regulators in different organs and will provide a powerful tool for plant physiology research.     

Arabidopsis ATG11, a scaffold that links the ATG1-ATG13 kinase complex to general autophagy and selective mitophagy

Autophagy is essential for nutrient recycling and intracellular housekeeping in plants by removing unwanted cytoplasmic constituents, aggregated polypeptides, and damaged organelles. The autophagy-related (ATG)1-ATG13 kinase complex is an upstream regulator that integrates metabolic and environmental cues into a coherent autophagic response directed by other ATG components. Our recent studies with Arabidopsis thaliana revealed that ATG11, an accessory protein of the ATG1-ATG13 complex, acts as a scaffold that connects the complex to autophagic membranes. We showed that ATG11 encourages proper behavior of the ATG1-ATG13 complex and faithful delivery of autophagic vesicles to the vacuole, likely through its interaction with ATG8. In addition, we demonstrated that Arabidopsis mitochondria are degraded during senescence via an autophagic route that requires ATG11 and other ATG components. Together, ATG11 appears to be an important modulator of the ATG1-ATG13 complex and a multifunctional scaffold required for bulk autophagy and the selective clearance of mitochondria.     

From Arabidopsis to cereal crops: Conservation of chloroplast protein degradation by autophagy indicates its fundamental role in plant productivity

Autophagy is an evolutionarily conserved process leading to the degradation of intracellular components in eukaryotes, which is important for nutrient recycling especially in response to starvation conditions. Nutrient recycling is an essential process that underpins productivity in crop plants, such that remobilized nitrogen derived from older organs supports the formation of new organs or grain-filling within a plant. We extended our understanding of autophagy in a model plant, Arabidopsis thaliana, to an important cereal, rice (Oryza sativa). Through analysis of transgenic rice plants stably expressing fluorescent marker proteins for autophagy or chloroplast stroma, we revealed that chloroplast proteins are partially degraded in the vacuole via Rubisco-containing bodies (RCBs), a type of autophagosomes containing stroma. We further reported evidence that the RCB pathway functions during natural leaf senescence to facilitate subsequent nitrogen remobilization into newly expanding leaves. Thus, our recent studies establish the importance of autophagy in biomass production of cereals.     

Apple BT2 protein negatively regulates jasmonic acid‐triggered leaf senescence by modulating the stability of MYC2 and JAZ2

Jasmonic acid (JA) is shown to induce leaf senescence. However, the underlying molecular mechanism is not well understood, especially in woody plants such as fruit trees. In this study, we are interested in exploring the biological role of MdBT2 in JA‐mediated leaf senescence. We found that MdBT2 played an antagonistic role in MdMYC2‐promoted leaf senescence. Our results revealed that MdBT2 interacted with MdMYC2 and accelerated its ubiquitination degradation, thus negatively regulated MdMYC2‐promoted leaf senescence. In addition, MdBT2 acted as a stabilizing factor to improve the stability of MdJAZ2 through direct interaction, thereby inhibited JA‐mediated leaf senescence. Furthermore, our results also showed that MdBT2 interacted with a subset of JAZ proteins in apple, including MdJAZ1, MdJAZ3, MdJAZ4 and MdJAZ8. Our investigations provide new insight into molecular mechanisms of JA‐modulated leaf senescence. The dynamic JA‐MdBT2‐MdJAZ2‐MdMYC2 regulatory module plays an important role in JA‐modulated leaf senescence.     

Phytohormones and microRNAs as sensors and regulators of leaf senescence: Assigning macro roles to small molecules

Ageing or senescence is an intricate and highly synchronized developmental phase in the life of plant parts including leaf. Senescence not only means death of a plant part, but during this process, different macromolecules undergo degradation and the resulting components are transported to other parts of the plant. During the period from when a leaf is young and green to the stage when it senesces, a multitude of factors such as hormones, environmental factors and senescence associated genes (SAGs) are involved. Plant hormones including salicylic acid, abscisic acid, jasmonic acid and ethylene advance leaf senescence, whereas others like cytokinins, gibberellins, and auxins delay this process. The environmental factors which generally affect plant development and growth, can hasten senescence, the examples being nutrient dearth, water stress, pathogen attack, radiations, high temperature and light intensity, waterlogging, and air, water or soil contamination. Other important influences include carbohydrate accumulation and high carbon/nitrogen level. To date, although several genes involved in this complex process have been identified, still not much information exists in the literature on the signalling mechanism of leaf senescence. Now, the Arabidopsis mutants have paved our way and opened new vistas to elucidate the signalling mechanism of leaf senescence for which various mutants are being utilized. Recent studies demonstrating the role of microRNAs in leaf senescence have reinforced our knowledge of this intricate process. This review provides a comprehensive and critical analysis of the information gained particularly on the roles of several plant growth regulators and microRNAs in regulation of leaf senescence.     

Proteomic analysis of residual proteins in blades and petioles of fallen leaves of Brassica napus

Brassica napus L. is an important crop plant, characterised by high nitrogen (N) levels in fallen leaves, leading to a significant restitution of this element to the soil, with important consequences at the economic and environmental levels. It is now well established that the N in fallen leaves is due to weak N remobilisation that is especially related to incomplete degradation of foliar proteins during leaf senescence. Identification of residual proteins in a fallen leaf (i.e. incompletely degraded in the last step of the N remobilisation process) constitutes important information for improving nutrient use efficiency. Proteome analysis of the vascular system (petioles) and blades from fallen leaves of Brassica napus was performed, and the 30 most abundant residual proteins in each tissue were identified. Among them, several proteins involved in N recycling remain in the leaf after abscission. Moreover, this study reveals that some residual proteins are associated with energy metabolism, protection against oxidative stress, and more surprisingly, photosynthesis. Finally, comparison of blade and petiole proteomes show that, despite their different physiological roles in the non‐senescing leaf, both organs redirect their metabolism in order to ensure catabolic reactions. Taken together, the results suggest that a better degradation of these leaf proteins during the senescence process could enable improvements in the N use efficiency of Brassica napus.     

Lipid Profiling Demonstrates That Suppressing Arabidopsis Phospholipase Dδ Retards ABA-Promoted Leaf Senescence by Attenuating Lipid Degradation

Senescence is the last phase of the plant life cycle and has an important role in plant development. Degradation of membrane lipids is an essential process during leaf senescence. Several studies have reported fundamental changes in membrane lipids and phospholipase D (PLD) activity as leaves senesce. Suppression of phospholipase Dα1 (PLDα1) retards abscisic acid (ABA)-promoted senescence. However, given the absence of studies that have profiled changes in the compositions of membrane lipid molecules during leaf senescence, there is no direct evidence that PLD affects lipid composition during the process. Here, we show that application of n-butanol, an inhibitor of PLD, and N-Acylethanolamine (NAE) 12∶0, a specific inhibitor of PLDα1, retarded ABA-promoted senescence to different extents. Furthermore, phospholipase Dδ (PLDδ) was induced in leaves treated with ABA, and suppression of PLDδ retarded ABA-promoted senescence in Arabidopsis. Lipid profiling revealed that detachment-induced senescence had different effects on plastidic and extraplastidic lipids. The accelerated degradation of plastidic lipids during ABA-induced senescence in wild-type plants was attenuated in PLDδ-knockout (PLDδ-KO) plants. Dramatic increases in phosphatidic acid (PA) and decreases in phosphatidylcholine (PC) during ABA-induced senescence were also suppressed in PLDδ-KO plants. Our results suggest that PLDδ-mediated hydrolysis of PC to PA plays a positive role in ABA-promoted senescence. The attenuation of PA formation resulting from suppression of PLDδ blocks the degradation of membrane lipids, which retards ABA-promoted senescence.