Chloroplast Recycling and Plant Stress Tolerance

Abstract

Plastids have emerged as pivotal regulators of plant’s response to biotic and abiotic stresses. Chloroplasts have the ability to synthesize a variety of pigments, secondary metabolites, and phytohormones which help plant cells to withstand adverse conditions. Further, plastids communicate with the nucleus and other cellular organelles for the acquisition of essential molecules to survive under unfavorable conditions. They act as environmental sensors which not only synthesize molecules for stress tolerance but also induce nucleus-encoded genes for stress resilience. Senescence is a key developmental process in this context and plays an important role in the release of essential nutrients. Chloroplast proteolytic machinery plays a crucial role in the degradation or remodeling of plastid proteins resulting in the generation of numerous endogenous peptides which are present in the plant secretome. Plastid chaperone system is also activated for the repair/refold of damaged proteins resulting in improved tolerance to stresses. Autophagy is a conserved process that involves large-scale breakdown of chloroplast through piecemeal degradation and chlorophagy. The piecemeal degradation occurs through Rubisco-containing bodies (RCBs) and senescence-associated vacuoles (SAVs), whereas chlorophagy targets chloroplasts as a whole. Though information about chloroplast recycling is limited, the present work provides a comprehensive review on chloroplast recycling and its role in stress mitigation and adaptation in climate change scenarios.

1. Introduction

Leaf senescence is a complex biological process characterized by the active breakdown of cellular macromolecules and the subsequent remobilization of their components. During this process, proteins like Rubisco and other chloroplast proteins undergo gradual degradation, serving as a vital source of nitrogen for recycling. This degradation is closely linked with a decline in photosynthetic activity [1]. Notably, under conditions of darkness-induced starvation, chloroplast proteins, including Rubisco, can be degraded with their carbon skeletons being utilized for respiration [2]. As the levels of these essential components diminish, chloroplasts undergo structural changes, eventually transitioning into gerontoplasts. In gerontoplasts, thylakoid membranes break down, plastoglobules accumulate, and the overall population of chloroplasts in the cell declines.

The vacuole within leaf mesophyll cells, constituting a substantial portion of the total cell volume, is rich in a diverse array of lytic hydrolases. A significant proportion of the proteolytic activity responsible for degrading Rubisco is localized within the vacuolar fraction. Notably, senescence-associated induction of various vacuolar cysteine proteases has been observed [3]. Previous studies have proposed that sequential degradation of chloroplasts within the vacuole serves as the primary pathway for chloroplast protein degradation during leaf senescence. In electron microscopy observations, chloroplasts were found to either reside within the vacuole or within tonoplast invaginations in mesophyll protoplasts from senescing wheat leaves, suggesting a mechanism resembling phagocytosis [1]. This process of delivering cytoplasmic components to the vacuole for degradation is now recognized as autophagy.

Interestingly, the decline in Rubisco protein levels occurs more rapidly than the decrease in the chloroplast population size during senescence. Furthermore, the reduction in major chloroplast proteins does not proceed uniformly; for instance, Rubisco declines more quickly than light-harvesting complex II (LHCII) [4]. Both of these proteins are primarily synthesized during leaf expansion and exhibit lower turnover rates thereafter. These observations suggest the existence of alternative pathways or mechanisms beyond whole chloroplast autophagy that contribute to Rubisco degradation. Consequently, the study of Rubisco degradation has largely focused on chloroplast proteases rather than autophagy [5]. Notably, several proteases within chloroplasts have crucial roles in chloroplast development and maintenance. Genome-wide studies have uncovered prokaryotic-origin proteases within chloroplasts, with ATP-dependent proteases such as Clp, FtsH, and Lon being prominent enzymes involved in gradual protein degradation into amino acids and oligopeptides [6]. While some of these proteases are likely to play pivotal roles in senescence-associated bulk degradation of chloroplast proteins, the exact mechanisms remain a topic of debate [7].

2. Chloroplast recycling

Chloroplasts serve as vital organelles of the plant cell performing photosynthesis as well as production and storage of an array of biomolecules and waste materials. Pigments and light-harvesting proteins make up a significant portion of their nitrogen and Rubisco content. In the advanced stages of aging, chloroplast numbers dwindle. Studies employing electron microscopy have hinted at an intriguing process – chloroplast recycling, the complete self-consumption of chloroplasts, in leaves undergoing senescence induced by aging as well as biotic or abiotic stresses [1].

2.1 Chloroplast recycling in senescence

Senescence, the final stage of leaf development before cell death, includes the disintegration of chloroplast as well as the loss of protein, nucleic acid, pigments, lipids, and polysaccharides (such as starch). Because of this seer degradation, chloroplast ultimately loses either all or a majority of their ability to photosynthesis. The senescence of leaves and other green plant organs results in the annual breakdown of millions of tons of chlorophyll and photosynthetic protein [8]. This results in a loss of photosynthetic capacity, releasing nutrients like nitrogen for re-use in other parts of the plant. During senescence, chloroplast loses their photosynthetic mechanism, and nutrients are redirected to young tissues and storage organs. For instance, nitrogen extracted from mature grains in field-grown rice may represent 60% of the nitrogen released by sensing leaves [9]. In some species, abscission marks the end of senescence, requiring precisely timed petiole senses. As leaves degrade, mesophyll tissue starts to lose its green color and turn yellow, or tissue starts to change color. This is caused by the preferential degradation of chlorophyll over carotenoids, as well as other factors and the synthesis of new compounds such as anthocyanins and phenolic [10]. While chloroplast changes early in senescence, they are last to collapse as the part of this developmental program [11]. Key characterization of chloroplast degeneration includes altered volume and morphology, a transition from ellipsoid to circular shape, and internal membrane reorganization. The three steps that Tamari et al. [12] determined to be the most significant ultrastructural alterations connected with the transformation from mature chloroplast to gerontoplast are thylakoid membrane breakdown, increased plastoglobulus size and number, and modification and disorderness of the plastid envelope. Senescence in chloroplasts in terms of pigment composition resembles with the chromoplasts, yet differs in development, division capacity, retention of the genome, and biosynthetic ability. Chromoplasts have their own DNA, produce carotenoid, are capable of division, and create iron or young chloroplast, but on the other hand, senescent chloroplasts only form from mature chloroplast, are unable to divide, have eliminated all metabolic activity, and do not have their own DNA [13]. The term “gerontoplasts” was introduced to highlight these distinctions. Detecting the exact transition from chloroplast to gerontoplasts is challenging due to difficulties in identifying the senescence onset. During senescence, the natural process of aging and degradation in plant chloroplast undergoes dynamic changes to adapt protein import and export mechanisms to cellular demands. During their light-induced differentiation, chloroplasts import pre-proteins and amino acids from the cytosol. Nuclear-encoded proteins are imported into chloroplast through membrane transport complexes, and protein import is crucial for the regulation of plant adaptation processes. Under adverse stress and environmental conditions including senescence, to ensure regular cell function, the damaged chloroplast or one of its specialized components needs to be dismantled quickly. Similar to this way, during leaf senescence, chloroplast also exports organic materials including proteins and amino acids [14].

2.2 Chloroplast recycling in stress tolerance

Plastids have emerged as pivotal regulators of plant’s response to biotic and abiotic stresses. Chloroplasts have the ability to synthesize a variety of secondary metabolites and phytohormones which help out plant cells to withstand adverse conditions. Further, plastids communicate with the nucleus and other cellular organelles for the acquisition of essential molecules for survival under unfavorable conditions.

Chloroplasts act as environmental sensors which not only synthesize molecules for stress tolerance but also induce nucleus-encoded genes to be activated and produce stress-resilient proteins. The MEcPP (methylerythritol cyclodiphosphate) is an important constituent of isoprenoid biosynthesis in plastids and also acts as a retrograde signal to activate the expression of stress-responsive nucleus-encoded genes. MEcPP was first identified by screening for mutants with an elicited expression of HPL (hydroxyperoxide lyase). HPL is a nucleus-encoded stress responsive gene but encodes for a plastid enzyme, which plays a fundamental role in the synthesis of jasmonic acid and other stress-related proteins.

Constitutive expression of HPL led to hyperaccumulation of methylerythritol cyclodiphosphate and regulation of a series of nuclear genes [15]. Likewise, PAP (phosphonucleotide 3′-phosphoadenosine 5′-phosphate) is another important plastid metabolite that has explored retrograde signals for stress tolerance in plants [16]. Plastidic SAL1 (chloroplast 3′ (2′), 5′ -bisphosphate nucleotidase) can dephosphorylate PAP to AMP (adenosine monophosphate). The activity of SAL1 is inhibited, and as a result, PAP accumulation is increased as PAP has been explored to play a crucial role in drought stress tolerance and in the regulation of a number of nucleus-encoded stress-responsive genes [17]. Chloroplasts are sensitive to high-temperature stress, and more than 200 genes have been found to be upregulated in response to heat stress. Plants also induce leaf chlorosis wherein the activity of chlorophyll degrading enzymes is increased during photosynthesis. This rapid degradation of chlorophyll, in response to heat stress, is linked with the activation of genes encoding for pheophytinase and chlorophyllase [18].

Senescence is a key developmental process that has effectively been utilized by the plants to cope with biotic and abiotic stresses. The first organelle to be degraded during senescence is chloroplast which contains more than 75% of the leaf nitrogen. Meanwhile, nucleus and mitochondria remain active and provide energy for the progression of senescence. Most of the nitrogen is in the form of proteins, so proteolysis is the core step for its remobilization particularly during the reproductive stage. The degradation of chloroplast components involves different vesicular pathways which can be dependent and/or independent of the ATG (autophagy-related genes). ATG (autophagy-related gene)-dependent degradation pathways involve RCB (RubisCO-containing bodies) and ATI-PS (ATG8-interacting protein 1-plastid associated bodies). Compared with RubisCO-containing bodies, ATG8-interacting protein does not require functional autophagic machinery. ATI1 interacts with thylakoid localized proteins (PsbS, LHCA4, APE1, and PrxA), whereas RCB transports stroma proteins [19]. Under stress conditions, ATG-dependent pathways help in the removal of damaged chloroplast contents resulting in accelerated senescence and increased sensitivity to abiotic stresses. Hence, these proteins play a crucial role in the adaptation to adverse climatic conditions [20].

Under stress conditions, intensive degradation or remodeling of plastid proteins results in the generation of numerous endogenous peptides which are present in the plant secretome. The stress may lead to an increase in misfolded plastidic proteins which activates the chaperone system for the repair/refolding of damaged proteins as well as induces proteases for the degradation of proteins unable to be refolded. Hence, activity of chloroplast proteolytic machinery plays a crucial role in stress tolerance. Nature has blessed plastids with highly specialized proteases, that is, MAPs (methionine aminopeptidases) for the repair and maturation of not only chloroplast-encoded proteins but also of the nucleus encoded proteins [21]. Hence, plants manage to withstand stresses (biotic and abiotic) through recycling of their chloroplasts which involves degradation of the misfolded proteins as well as the repair/refolding of the damaged proteins [22].

3. Recycling and repair pathways

There are two pathways of plastid recycling and repair, that is, piecemeal degradation where chloroplasts are degraded gradually, while the other pathway is known as chlorophagy in which the whole chloroplast is degraded for recycling.

3.1 Piecemeal degradation of chloroplasts

In the process of piecemeal degradation and chloroplast recycling, a specific mechanism for releasing Rubisco from chloroplasts and subsequently breaking it down in other cell compartments has been proposed as an explanation for the early decline in Rubisco levels compared to the chloroplast population. As previously summarized [23], ATG-dependent autophagy via Rubisco-containing bodies (RCBs), which are sent to the central vacuole for degradation, and an ATG-independent alternative pathway involving senescence-associated vacuoles (SAVs) are currently evident to be at least two distinct transport pathways responsible for breaking down stromal proteins outside the chloroplast.

3.1.1 Rubisco-containing bodies (RCBs)

The RCBs were initially discovered in naturally aging wheat leaves (T. aestivum) [4]. According to thorough immunoelectron-microscopic investigations, Rubisco is occasionally contained within tiny spherical structures (RCBs), which are mainly found in the cytoplasm and occasionally in the vacuole. The stromal protein Gln synthetase is found in RCBs, which also have an electron density like that of chloroplast stroma. Notably, RCBs lack major membrane proteins including cytochrome f, LHCII, and the -subunits of ATPase coupling factor 1 as well as thylakoid structures. The isolation membranes (phagophores) that surround the double membranes of RCBs in the cytoplasm are thought to be derived from the chloroplast envelope. In the early phases of leaf senescence, when Rubisco levels begin to decline but chlorophyll content stays largely constant, RCBs are frequently observed.

The phenomenon of gradual chloroplast breakdown through RCBs (Rubisco-containing bodies) seems to be prevalent across various plant species, serving as a crucial mechanism for recycling proteins during both growth and in response to environmental challenges. The RCBs have also been identified in young tobacco leaves (Nicotiana tabacum), where they are known as Rubisco-vesicular bodies (RVBs), and in rice leaves (Oryza sativa) exposed to salt stress conditions. Interestingly, the RCBs found in rice leaves under salt stress exhibit distinctive structural features compared to previously documented RCBs. These rice RCBs possess inner membrane structures, potentially formed from vesicles originating from invaginations of the chloroplast’s inner envelope. Furthermore, they occasionally contain crystalline inclusions that develop within chloroplasts under osmotic stress and disappear during the recovery phase. It is plausible that RCBs play a role in breaking down these crystalline inclusions, and the possible mechanisms involved in RCB formation during salt stress have been extensively detailed [24].

The connection between RCBs (Rubisco-containing bodies) and autophagy has been unveiled in Arabidopsis through the use of reverse genetics and live-cell imaging techniques. In this process, RCBs can be visualized by employing stroma-targeted green fluorescent protein (GFP) (or RFP) or a fusion involving a small subunit of Rubisco (RBCS) with GFP (or RFP). This fusion interacts with the plant’s endogenous large Rubisco subunits (RbcL) and RBCS molecules, resulting in the creation of Rubisco-GFP (or RFP) [25]. When Arabidopsis leaves expressing these fluorescent markers are treated with concanamycin A, which suppresses vacuolar lytic activity, spherical structures displaying GFP or RFP fluorescence, devoid of chlorophyll fluorescence, appear within the vacuolar lumen. These structures are referred to as RCBs. Additionally, the accumulation of RCBs is disrupted in autophagy-defective mutants known as atg5 and atg4a4b [25, 26]. In normal, wild-type cells, stroma-targeted RFP and the GFP-ATG8a fusion, serving as a marker for autophagosomes and autophagic bodies, are observed together within autophagic bodies located in the vacuole [27]. These findings provide evidence supporting the notion that RCBs constitute a distinct type of autophagic body containing Rubisco and potentially other proteins localized within the stroma.

3.1.2 Senescence-associated vacuoles (SAVs)

A novel type of diminutive lytic vacuole, known as the senescence-associated vacuole (SAV), has come to light within the cytoplasm of aging leaves in plants like soybean (Glycine max), Arabidopsis, and tobacco. This discovery has unveiled an alternative route for the breakdown of chloroplast proteins, distinct from the conventional chloroplast-based degradation [28]. Remarkably, SAVs exclusively emerge in photosynthetic tissues undergoing senescence, harboring a unique senescence-specific cysteine-protease, SAG12 (senescence-associated gene 12).

Comparable to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) containing bodies (RCBs), SAVs encompass stromal proteins like Rubisco and glutamine synthetase, and stromal-targeted GFP. However, they lack thylakoid proteins such as the LHCII. Additionally, unlike RCBs, SAVs are laden with chlorophyll a. Intriguingly, there is no substantiated evidence thus far to suggest that SAVs engage autophagic machinery. Notably, it was postulated that SAVs still manifest even in the absence of autophagy, as demonstrated in the autophagy-defective atg7 mutant, albeit supporting data were conspicuously absent in the original study [29]. This underscores a semblance in the content of RCBs and SAVs, while their formation mechanisms remain unequivocally distinct. A recent investigation unveiled that SAV buildup coincides with the induction of autophagy in the Arabidopsis des1 mutant, which lacks L-cysteine desulfhydrase 1, an enzyme crucial for cysteine degradation [30]. However, further research is imperative to elucidate the intricate interplay between ATG-dependent autophagy and SAVs.

The visual contrast between SAVs (stroma-associated vacuoles) and RCBs (Rubisco-containing bodies) is quite remarkable and can be easily appreciated under the microscope. The RCBs are normally found enclosed by a double membrane and display internal electron density similar to that of the chloroplast stroma. In sharp contrast, SAVs are enclosed by a single membrane. The electron density within SAVs resembles that of the central vacuole and is notably lower than that found in the chloroplast stroma. Additionally, SAVs often contain dense aggregates within their interior, potentially composed of partially degraded cellular components.

The exact mechanism by which stromal proteins are directed to SAVs remains a mystery. As previously discussed in a comprehensive review [31], one possible pathway involves stromal proteins initially traversing the chloroplast envelope before being transferred directly to SAVs through a yet-to-be-revealed mechanism. Alternatively, similar to the process of piecemeal microautophagy of the nucleus in yeast, it is plausible that SAVs themselves encapsulate a portion of the chloroplast or stromule, forming a structure similar to RCBs. The SAVs exhibit a higher level of acidity compared to the central vacuole and possess potent proteolytic capabilities. Thus, chloroplast particles enclosed within SAVs would undergo rapid degradation, resembling the fate of RCBs within the central vacuole. It is important to note that this phenomenon can only be observed in the presence of concanamycin A.

3.1.3 Protein import/export and chloroplast recycling

The majority of chloroplast proteins originate from nuclear genes and are guided into chloroplast by TOC (translocon at the outer chloroplast membrane) and TIC (translocon at the inner chloroplast membrane) complexes under normal conditions. The process of transporting these nuclear-encoded proteins into the chloroplast is a highly orchestrated series of steps that rely on specific molecules and energy sources. These proteins contain a distinct molecular tag called a transit peptide, which acts as the navigation signal, directing them to the appropriate location within the chloroplast. Several key stages are involved in the import of protein: firstly, they are identified by transit peptide specialized proteins called cytosolic chaperones and targeting factors. These molecules create a protective complex around the protein, preventing it from folding prematurely and maintaining it in a state ready for import. After identification and binding, the protein-chaperon complex intratas with molecular machinery situated at the outer membrane of the chloroplast, referred to as the TOC complex [32]. This nitration triggers the movement of the protein across the outer chloroplast membrane. Upon successfully crossing the outer membrane, the protein chaperon complex encounters another translocon, the TIC complex, embedded in the chloroplast inner membrane [33]. This complex assists in transporting the protein across the inner membrane, ensuring its access to the inner space of the chloroplast. Once the protein has reached the inner space of the chloroplast, the transit peptide is cleaved off. This step is vital for the protein to adopt its final functional shape. Furthermore, molecular chaperones within the chloroplast environment aid in correctly folding the protein, ensuring its effective participation in the intricate biochemical process of organelles. In essence, the process of chloroplast protein import involves a sequence of precisely coordinated events, safeguarding the integrity and functionality of the transported proteins within the dynamic environment. While most proteins are imported, there are situations when specific proteins need to be exported. Some of these exported proteins play roles in photosynthesis, plastid division, or inter-organelle signaling. The export mechanism typically involves recognizing sorting signals and their integration with the export machinery [34].

A controlled physiological process is involved in the gradual breakdown of cellular components leading to significant alterations in chloroplasts’ structure and function, thereby influencing the intricate protein import and export process. Prominent observations highlight the changes that take place in such process during senescence. Firstly, studies indicate that the rates of protein import into chloroplast vary as senescence advances. These changes often connect to modifications in the expression of essential components in the TOC and TIC complexes, which play vital roles in facilitating efficient protein translocation across the chloroplast membrane [35]. As chloroplast age during senescence, the importance of quality control mechanisms becomes more prominent. These mechanisms identify and transport mis-folded protein out of the chloroplast to prevent their buildup and potential damage. This involves the retrograde transport of malfunctioning proteins to the cytoplasm for degradation. Senescence introduces distinct sorting signals known as senescence-associated sorting signals. These signals guide proteins to specific locations within the chloroplast, highlighting the unique nature of protein transport during senescence. Moreover, senescence stimulates substantial shifts in gene expression and signal transduction pathways [36]. Regulatory proteins managing this process might need to be exported from chloroplast toward organelles like vacuoles and nucleus. This export plays a vital role in coordinating the different events associated with senescence.

In the process of chloroplast protein degradation, two vesicles’ categories play vital roles: Rubisco-containing bodies (RCBs) and senescence-associated vacuoles (SAVs) (Figure 1). The RCBs are smaller, sphere-shaped vesicles, and enclosed by dual membranes that consist of Rubisco, excluding thylakoid proteins. They are considered as the autophagosomic bodies responsible for transporting stomata proteins toward the vacuoles [37]. Notably, RCBs operate during darkness, senesce, and leaf carbon homeostasis, participating in Rubisco reduction under stall stress in soybean [38]. In contrast, SAVs are characterized by elevated proteolytic and acidic activity compared to central vacuole. These vessels are prominent during senescence with chloroplast-containing cells, notably accumulating in senescence leaves. They facilitate the breakdown of soluble photosynthesis proteins within the chloroplast stroma, including glutamine synthetase and a large unit of Rubisco. Unlikely autophagy, SAVs can specifically degrade chloroplast components, yet the mechanism of their formation and translocation of chloroplast components to SAVs remains unknown [39].

Figure 1.

Cellular pathways involved in chloroplast protein import showing protein trafficking. SAVs represent senescence-associated vacuoles, RCBs are for rubisco-containing bodies, and chloroplast-containing vesicles (CCVs) are for CV-containing vesicles.

Recent research has uncovered that SAVs are implicated in the chlorophyll, and PSI degradation throughout senescence, supported by their strong cysteine protease activity, and the presence of the senescence-specific protease perhaps contribute to the breakdown of the Rubisco during leaf senescence [40]. These vesicles are crucial for the proteolysis of chloroplast proteins. Chloroplast proteins need to be moved outside of the plastid for the previously mentioned pathways to function [41]. Lately, a significant gene chloroplast vesiculation (CV) has been recognized, involving translocation of chloroplast proteins pathway external to plastid. This gene’s expression has been shown to elevate during ordinary and abiotic stress induced senescence. Once chloroplast proteins enter the plastids, CV destabilizes the chloroplast, prompting the formation of CCVs during senescence, which subsequently deliver chloroplast proteins to the vacuoles for proteolysis [42]. The CV protein interrelates with several chloroplast proteins, primarily thylakoid components, in plastids, where it localizes. Intra-plastidic vesicles, or CCVs, are produced as a result of the CV gene. These CCVs eventually die from the plastid and travel to the central vacuole, carrying proteins from stromal and thylakoid membranes. Importantly, this degradation process through CCVs operates independently of autophagy and SAVs. The CV protein interacts mainly with thylakoid components, triggering the development of vesicles. The RD26 transcription factor has recently been linked to the induction of the CV-related gene expression. The RD26 regulates the direct degradation of chloroplast proteins during senescence [43]. As chlorophyll breakdown initiates within chloroplasts in senescent leaves, non-fluorescent chlorophyll proteins are released and ultimately broken down in vacuoles. This suggests that senesces or stress can stimulate the degradation of chloroplast protein, even the entire chloroplast, leading to peptide accumulation within the chloroplast, the cytoplasm, and various vesicles. Notably, these peptides could potentially be transported into the extra-cellular space through vesicles and vacuole fusion, aided by outer and inner peptidases, as well as peptide and amino acid transporters.

3.2 Role of autophagy in chloroplast recycling

Autophagy is a highly conserved biological mechanism in eukaryotic organisms. This process involves the engulfing of cellular components within membrane-bound vesicles, followed by their subsequent degradation through lytic processes. Whole organelles can also be enclosed within these membrane structures as part of autophagy [44]. These cellular components wrapped in membrane-bound vesicles are taken to special compartments called vacuoles or lysosomes. Inside these compartments, enzymes called hydrolases break down both the vesicles and their contents. There are two different forms of autophagy, known as micro-autophagy and macro-autophagy, which have been seen in various organisms, including plants [45]. In micro-autophagy, parts of the cell’s fluid are directly taken in by the folding of the vacuolar membrane. In the more common form called macro-autophagy, often just referred to as autophagy, the cell’s content is enclosed within a double-layered structure called an autophagosome. The fusion event between the outer membrane of the autophagosome and the vacuolar membrane facilitates the transfer of the enclosed inner membrane-defined structure, known as the autophagic body, into the interior space of the vacuole.

In more recent times, a breakthrough occurred with genome-wide studies, which paved the way for a molecular examination of plant autophagy. These studies identified numerous genes in plants that are similar to yeast ATGs and also explored the effects of disrupting these genes in Arabidopsis (Arabidopsis thaliana). Researchers have explored the roles of Arabidopsis ATGs by utilizing T-DNA insertional knock-out mutants and RNA interference knockdown mutants. Additionally, they have established a live-cell system for monitoring autophagy in plants. This system involves the use of a GFP fused with ATG8, which acts as a marker to track the presence of autophagosomes. These molecular methods have demonstrated the crucial function of plant autophagy in response to food deficiency, abiotic stressors, and pathogen infection. They have also confirmed that the central autophagy apparatus functions in plants but not in yeast [46]. Additionally, it has been demonstrated that target of rapamycin (TOR) kinase functions as a negative regulator in yeast and Arabidopsis [47].

Plant autophagy was initially studied for its ability to respond to nutrient deprivation, a critical role similar to that of yeast. Autophagy is triggered when cultured plant cells lack externally supplied sucrose. During this period, key ATGs linked to Ub-like conjugation systems experience temporary increases in activity [48]. Scientists have examined the role of autophagy in nutrient recycling by studying autophagy-deficient (atg) mutant plants. Essentially, Arabidopsis atg mutants can complete their life cycles [1].

However, they struggle to survive extended periods of nitrogen and/or carbon starvation. Additionally, they exhibit accelerated leaf aging and cell death, along with reductions in chlorophyll and photosynthetic proteins, even when nutrient and growth conditions are favorable. This premature aging affects seed production, leading to lower yields in atg mutants. Consequently, it was previously concluded that while autophagy plays a vital role in nutrient recycling during starvation and senescence in plants, it does not primarily target chloroplasts, despite their abundance in leaf degradation [45].

3.3 Chlorophagy: autophagy of whole chloroplasts

In the advanced stages of aging, chloroplast numbers dwindle. Studies employing electron microscopy have hinted at an intriguing process – the complete self-consumption of chloroplasts, an occurrence termed “chlorophagy,” in leaves undergoing senescence induced by darkness [1]. In the world of Arabidopsis, a plant’s individual leaf darkened for experimentation experiences a rapid onset of senescence. Remarkably, within a few short days, both the quantity and size of chloroplasts within these leaves diminish significantly. However, in the case of individually darkened leaves (IDLS) from the atg4s mutant, senescence, evident as chlorosis, unfolds much like in the wild-type, yet the drop in chloroplast count and to some extent, their size, is hindered [49].

What’s more, beyond the realm of conventional chloroplast behavior, small chloroplasts retaining their chlorophyll fluorescence come into view inside the vacuole after 3 days of the IDL treatment in wild-type specimens, but this phenomenon remains absent in the atg4 mutant. Given that the formation of Rubisco-containing bodies (RCBs) consumes elements from both the stroma and the chloroplast envelope, it is a logical assumption that this process contributes to the shrinking of chloroplasts. These shrunken chloroplasts, reminiscent of gerontoplasts, are then ferried into the vacuole through the machinery of autophagy.

Notably, mutations that disrupt the normal functioning of chloroplasts can trigger the wholesale degradation of these organelles, a process likely attributable to chlorophagy. This degradation appears to occur independently of senescence or starvation. For instance, partially degraded chloroplasts make their home within the vacuoles of cotyledon cells belonging to the Arabidopsis ppi40 mutant, a genetic variant deficient in a protein homologous to the 40 kDa protein found in the inner envelope membrane of chloroplasts’ translocon (Tic complex). In the advanced stages of aging, chloroplast numbers dwindle. Studies employing electron microscopy have hinted at an intriguing process – the complete self-consumption of chloroplasts, an occurrence termed “chlorophagy,” in leaves undergoing senescence induced by darkness [1]. As chloroplast transfer to the vacuole is observed even under well-fed conditions, it is posited that plants utilize autophagy to eliminate defective plastids, ensuring quality control of their organelles.

The Arabidopsis mex1 mutant, which lacks the maltose transporter in the chloroplast envelope, presents a fascinating scenario. This mutant accumulates higher-than-normal levels of maltose and starch within its chloroplasts and displays a chlorotic phenotype even when it’s not undergoing senescence. What’s particularly intriguing is that various components of chloroplasts, such as thylakoid membranes, starch granules, and plastoglobules, have been found inside the vacuole of the mex1 mutant [50]. These discoveries have given rise to a hypothesis suggesting that the increased maltose concentration in the mex1 mutant disrupts chloroplast function, possibly setting off a form of retrograde signaling that initiates the degradation of chloroplasts.

4. Conclusions

Green photosynthetic organisms including plants, algae, and cyanobacteria are the source of energy and carbon fixation through photosynthesis. Green pigments containing plastid “chloroplasts” are the hub of photosynthesis. It supplies energy to all living organisms. Photosynthesis products, through various biochemical pathways, produce numerous macromolecules. Moreover, chloroplasts become an abundant source of necessary organic nitrogen and other nutrients through the process of chloroplast recycling in senescing leaves. Such nutrients are utilized in growth, reproduction, storage organs, and new plant organs. The protein translocation mechanism is essential for chloroplast function and plays a critical role in cellular adaptations during senescence. The dynamic changes observed in these processes during senescence highlight their significance in plant adaptation to changing environmental conditions and developmental stages. Though limited literature is available, a deep understanding of the protein import/export during senescence and/or chloroplast recycling can potentially lead to the progression of strategies to enhance crop productivity and stress tolerance, contributing to agriculture sustainability and food security.