KWARA STATE UNIVERSITY, MALETE

The Green University for Community Development and Entrepreneurship

FACULTY OF PURE AND APPLIED SCIENCE

A SEMINAR REPORT ON

AUTOPHAGY

BY

Beloved Kristiloluwa AJANI

(20D/57BC/01394)

i
 A SEMINAR REPORT ON

AUTOPHAGY

BY

Beloved Kristiloluwa AJANI

(20D/57BC/01394)

A REPORT SUBMITTED TO THE DEPARTMENT BIOCHEMISTRY,

FACULTY OF PURE AND APPLIED SCIENCES, KWARA STATE UNIVERSITY,

MALETE.

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD

OF BACHELOR OF SCIENCE (B.Sc. DEGREE IN BIOCHEMISTRY)

MAY, 2023.

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 DECLARATION

I hereby declare that this seminar report has not been submitted by other person for any

degree or qualification at higher institution. I also declare that the information provided

therein are mine and those that are not mine are properly acknowledged.

………………………… ……………………………

Name of Student Signature and date

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 CERTIFICATION

This is to certify that this report was written by Beloved Kristiloluwa Ajani

(20D/57BC/01394) of the Department of Biochemistry, Faculty of Pure and Applied

Sciences, Kwara State University, Malete. The report has been read and approved as meeting

part of the requirements for the award of Bachelor Science (B.Sc. Hons) Degree in

Biochemistry of Kwara State University, Malete.

....................................... ………………………..

Mrs. L. A. Usman Date

(Supervisor)

………………………….. …………………………..

Dr. R. A. Aladodo Date

(Departmental coordinator)

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 DEDICATION

This seminar work is dedicated to Almighty God, the author and finisher of faith. I am

grateful and thankful for his many blessings, wisdom and guidance. He has been the source

of strength throughout the period of my work and only on His wings have I soared.

I also dedicate this work to my parents Professor and Dr (Mrs) E.O Ajani. I thank them for

their input and encouragement every step of the way. May they reap the fruits of their labour.

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 ACKNOWLEDGEMENT

I use this medium to acknowledge my supervisor, Mrs. L.A Usman, for her guidance and

impact through the course of this seminar work. Thank you, ma’am, for helping to mold and

shape my journey in this period of study. May God repay you beyond measure.

I Thank the Departmental coordinator, Dr. Mrs. Raliat A Aladodo and other member of

academic staff of the Department of Biochemistry KWASU including Dr. Mutiu Alabi, Dr.

Rasheed B Ibrahim, Dr. Sulyman A Olanrewaju, Dr. Mrs. Asiat Ba’Allah for their guidance

and support in the pursuit of my academic programme.

I am grateful to my friends for their advice and for their friendship in the course of putting up

this report.

Finally, my special appreciation goes to my siblings, Precious Kristidamilola and Promise

Kristilere, for their support and for being sources of inspiration. God bless all you do.

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 TABLE OF CONTENT

Front Page…...………………………………………………………………………………. i

Title Page…..………………………………………………………………………………...ii

Declaration…………………………………………………………………………………...iii

Certification…………………………………………………………………………………. iv

Dedication…………………………………………………………………………………….v

Acknowledgement……………………………………………………………………………vi

Table of content………………………………………………………………………………vii

List of Tables…………………………………………………………………………………ix

List of Figures…………………………………………………………………………………x

CHAPTER 1: INTRODUCTION………………………………………………………….1

CHAPTER 2: BIOCHEMISTRY OF AUTOPHAGY

2.1 Mechanism of Autophagy…………………………………………………………....5

2.2 Signaling Pathway and Regulation…………………………………………………...6

2.3 Types of Autophagy………………………………………………………………….8

2.4 Selective Autophagy………………………………………………………………….11

2.5 Interplay of Autophagy and Reactive Oxygen Species………………………………12

CHAPTER 3: AUTOPHAGY IN HUMAN HEALTH

3.1 Physical factors that can induce Autophagy………………………………………….15

3.2 Effect of Autophagy on T cell function………………………………………………16

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3.3 Role of Autophagy in healthy aging………………………………………………….18

3.4 Autophagy in neurodegenerative diseases…………………………………………...19

3.5 Autophagy in cancer………………………………………………………………….23

3.6 Autophagy in cellular death………………………………………………………….25

CHAPTER 4: AUTOPHAGY IN PLANTS

4.1 Autophagy in Vegetative Growth…………………………………………………….28

4.2 Role of Autophagy in Leaf Senescence……………………………………………...29

4.3 Role of Autophagy in Nutrient Availability………………………………………….30

4.4 Autophagy in Response to Abiotic Stress……………………………………………34

4.5 Autophagy in Response to Biotic Stress……………………………………………..35

CONCLUSION………………………………………………………………………………37

REFERENCES……………………………………………………………………………….38

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 LIST OF TABLES

Table 1.1 Major Mammalian Autophagic Proteins

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 LIST OF FIGURES

Figure 1 Stages involved in Autophagy mechanism

Figure 2 Schematic diagram of types of autophagy in mammalian cells

Figure 3 Schematic diagram of macroautophagy

Figure 4 Role of autophagy in various diseases and disorders

Figure 5 Types of Autophagy found in plants

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 CHAPTER ONE

INTRODUCTION

Autophagy is derived from the Greek words “auto” meaning self and “phagy” meaning

eating. It is a natural cellular mechanism by which cells in the body degrade unnecessary or

damaged components within the cell. Autophagy is an evolutionarily conserved process that

occurs ubiquitously in all eukaryotic cells (Reggiori and Klionsky, 2002). It is an important

process for degrading proteins and organelles (via vacuole in yeast and plants or via lysosome

in animals) to facilitate intracellular recycling (Marshall and Vierstra, 2018).

Autophagy is a natural regulatory mechanism which retains beneficial substances and at the

same time removes harmful substances from body, eradicates damaged organelles, proteins

and cancerous materials and eliminate foreign pathogens such as viruses via a degradative

lysosomal pathway. The concept of autophagy emerged in the 1950s when Christian de Duve

reported existence of various hydrolases leading to discovery of lysosome as cellular

organelle. This discovery was aided with the discovery of the electron microscope. In 1963,

he termed autophagy as a phenomenon during which cells fuse protein containing vesicles

(autophagosomes) with lysosomes which leads to the decomposition of cellular protein.

In current science, autophagy is responsible for removal of long-lived proteins, damaged

organelle and malformed proteins during biosynthesis by lysosome (Baehrecke, 2005).

Through this process, autophagy joins many other organs and processes facilitating

homeostasis. While self-eating sounds harmful, its action helps to get rid of toxic materials in

the cells and repair them. The process is meant for regulating diverse cellular functions

including; growth, differentiation, response to nutrient deficit and oxidative stress, cell death,

macromolecule and organelle turnover.

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In areas such as health, illness, infection, degeneration, and genetic or lifestyle-related

disorders, the functions of autophagy have been investigated. Different infectious pathogens

have been made more contagious in the modern age by causes like globalization, open trade,

climate change, population booms, public health failures, immunological stresses and

mutations, species hopping, and increasing drug resistance in viruses. Recent studies have

revealed that autophagy plays a crucial role in a number of pathogenic viral and bacterial

illnesses that are posing serious dangers to humans. Studies have shown that there has been

an increase in the incidence of lifestyle and genetic diseases, such as cancers and

neurodegenerative disorders (Alzheimer’s, Parkinson’s, and Huntington’s diseases), which

affect the quality of life (Rekha et al., 2019).

Though overcoming these challenges has been overwhelming, advances in science and

technology have contributed in no small measure towards eradicating them. Through Science

and Technologies, novel, alternative, and complementary therapeutic options have been

developed, including phages, homing peptides, cytokines, siRNA, viral inhibitors, Toll-like

receptors (TLRs), antibodies, probiotics, herbs, phytomedicines, nanomedicines, and

immunomodulatory techniques (Lee et al., 2018; Rekha et al., 2019)

Autophagy is the first mechanism to clear endogenous debris and exogenous substances and

maintain normal physiological conditions in all eukaryotic cell. Besides maintaining

homeostasis, autophagy also regulates the development, differentiation, and maturation of

cells, such as endothelial cells, erythrocytes, and adipocytes. These cells are involved in

normal physiological (e.g., erythrocytes in respiration), immunological (e.g., mononuclear

cells in immunity), metabolic (e.g., adipocytes in fat metabolism), growth (e.g., osteocytes in

bone growth), and development (e.g., spermatozoa or ova in reproduction) processes.

Autophagy is also involved in clearing abnormal protein accumulations and correcting

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mitochondrial disorganization. Autophagy might play both physiological and pathological

roles since it is involved in overcoming cell stresses (Tong et al., 2020).

Autophagy have been shown to be induced by several stimuli including stress, amino acid

starvation (Battu et al., 2018), rapid declines in trophic factors or hormones (such as sex-

based differences) (Congdon, 2018), lipid starvation (Joy et al., 2018), impaired intracellular

cholesterol trafficking (Arenas et al., 2017), protein products, and infectious pathogens (Lee

et al., 2018). These stimuli can affect the autophagic function and induce different

morphological consequences via diverse signaling pathways; for instance, suppressing

phosphatidylinositol-3-kinase (PI3K) inhibitors and Beclin 1 inhibits the starvation-induced

mitochondrial autophagy, but not the neurotoxin (1-methyl-4-phenylpyridinium)-mediated

autophagy (Zhu et al., 2007; Li et al., 2016).

Although autophagy was discovered over 50 years ago, its molecular mechanisms were only

understood in the late 1990s following a genetic screening in yeast, which revealed mutations

in autophagy-related genes. At least 30 yeast autophagy genes (Atgs) have been identified,

many of which have mammalian cell homologs as depicted in Table 1 (Mizushima et al.,

2008; Ravikumar et al., 2010).

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Table 1: Major mammalian Autophagic Proteins

Source: Mizushima et al., (2008); Ravikumar et al., (2010).

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 CHAPTER 2

BIOCHEMISTRY OF AUTOPHAGY

2.1 Mechanism of Autophagy

The process of autophagy can be broken down into 5 stages; initiation, nucleation,

elongation, fusion and degradation (Figure 1). Autophagy involves formation of a double-

membrane vesicle, which confines cytoplasm, malformed proteins, long-lived proteins and

organelles thereafter binding with lysosomes for degradation. When autophagy is induced, an

isolation membrane called phagophore is formed which expands to engulf intra cellular cargo

thereby sequestering the cargo in a double membraned autophagosome (Mizushima, 2007).

Phagophore biogenesis requires a set of proteins called core autophagy machinery

(Mizushima et al., 2011; Wen and Kilonsky, 2016). The autophagosome fuses with a

lysosome or vacuole to decompose cytoplasmic contents it has taken up (Mizushima and

Komatsu, 2011). Formation of this double membrane vesicle is a complex process involving

16 autophagy related proteins and 2 ubiquitin like conjugation system;

i. Microtubule-associated protein light chain 3 (LC3) binding reacting system; ubiquitin

like protein LC3 is involved in elongation and closure of the autophagosome by

binding to phosphophatidylethanolamine (PE).

ii. Atg 12-Atg 5 binding reaction system; ubiquitin like protein Atg12 covalently binds

to Atg5 via Atg7 and Atg10. The system then reacts and binds to Atg16L1 (Lystad et

al., 2019).

The next step is the fusion of mature autophagosome with the specialized endosomal

compartment that is the lysosome to form the autolysosome (Mizushima, 2007).

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Figure 1: Stages involved in Autophagy Mechanism

Source: Mizushima et al. (2011).

2.2 Signaling Pathway and Regulation

Signaling pathway describes a series of chemical reactions in which a group of molecules in a

cell work together to control a cell function. Given the important role autophagy plays in the

maintenance of cellular homeostasis and survival under stress conditions and its involvement

in various aspects of animal development and pathophysiology, there is need for autophagy to

be finely regulated to avoid either excessive or insufficient activity (Yin et al, 2016).

Two signaling cascades that sense nutrient status, activate cell division, and regulate

autophagy are the Target of Rapamycin Pathway (TOR) and cAMP-PKA (cyclic Adenosine

Monophosphate-Protein Kinase A) pathways.

2.2.1 Nitrogen-dependent regulation/ TOR pathway

Long before the discovery of the Atg genes, it was recognized that a lack of glucose or amino

acids would cause autophagy (Deter and de Duve, 1967). The primary sensor of amino acid

and nitrogen change is TOR or the mammalian homolog MTOR (Mechanistic Target of

Rapamycin). Autophagy is initiated by the down-regulation of the TOR complex as a

6
consequence of various stimuli, such as developmental and nutritional signals (Marshall and

Vierstra, 2018). TOR/MTOR is an essential serine/threonine kinase belonging to the

phosphatidylinositol kinase-related kinase family and negatively regulates autophagy (Díaz-

Troya et al., 2008). It senses and integrates multiple environmental signals to inhibit

catabolism and coordinate cell growth. MTORC1 can be activated by energy status, nutrient

levels, growth factors and amino acids (Laplante and Sabatini, 2012).

For yeast in nutrient-rich conditions, TORC1 acts by directly or indirectly causing hyper

phosphorylation of autophagy protein Atg 13. TORC1 directly phosphorylates Atg13, Atg1

and Atg14 thereby preventing formation /activation of the Atg1-Atg13-Atg17-Atg31-Atg29

complex (Kamada et al., 2010) which suppresses autophagy-specific Ptdlns3k, thus inhibiting

autophagy induction (Yuan et al.,2013).

In mammalian cells, amino acids are sensed by the vacuolar-type H + translocating ATPase,

which is present in the lysosome membrane, in conjunction with RRAG proteins and the

Ragulator complex (Sancak et al., 2010) which coordinately direct MTORC1 to the lysosome

membrane where it becomes activated by GTPase RHEB (Bar-peled et al., 2012). Reduced

TOR activity induces autophagy, again ensuring that the cell adapts to its changing

environment through reduced growth and increased catabolism

2.2.2 Energy/glucose-dependent regulation/cAMP-PKA pathway

Regulation of autophagy by glucose metabolism and energy level is vital for cellular

homeostasis. In the presence of glucose, PKA is activated by binding with cAMP. PKA then

phosphorylates Atg1 and Atg13, which prevents localization of Atg13 to the Phagophore

Assembly Site (PAS) (Stephan et al., 2009). In addition, PKA can inhibit autophagy by direct

phosphorylation of TORC1 or in mammalian cells by indirect activation of MTORC1

through inhibition of AMPK. AMPK is the major energy sensor in the cell activated by

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increased AMP: ATP ratio, which is one of the outcomes of glucose depletion or other types

of stress such as mitochondrial dysfunction (Hardie et al., 2012). When AMPK senses low

energy, it promotes autophagy by inhibiting MTOR activity through direct negative

phosphorylation of RPTOR/raptor (sub-unit of MTORC1 complex) or phosphorylation and

activation of TSC1/2 complex (negative regulator of MTORC) (Russel et al., 2014)

2.3 Types of Autophagy

Autophagy is a lysosome-mediated degradative process of eukaryotic cells to digest their own

constituents during development or starvation. It is broadly divided into three types;

macroautophagy, microautophagy and chaperon mediated autophagy (Figure 2) and while

each is morphologically distinct, all three lead to the delivery of cargo to the lysosome for

degradation and recycling (Yang and Kilonsky, 2010).

Figure 2: Schematic diagram of types of autophagy in mammalian cells

Source: Yang and Kilonsky, 2010

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2.3.1 Macroautophagy

Macroautophagy is the most studied route of autophagy and as such, macroautophagy is often

times referred to as autophagy. Macroautophagy involves the formation of subcellular

double-membrane-bound structures called autophagosomes which contain degradable

contents to sequester cytoplasmic materials, and deliver them into lysosomes for breakdown

by lysosomal enzymes (Lawrence and Zoncu, 2019). The recycling of substrates through

macroautophagy plays an important role in maintaining cellular homeostasis (Dikic and

Elazar, 2018; Levine and Kroemer, 2019). In macroautophagy, a cup-shaped double-

membrane structure, named the phagophore, is formed at the phagophore assembly site

(PAS). Phagophore arises from endoplasmic reticulum (ER), mitochondria and plasma

membrane or from ER-mitochondria contact sites (Zhuang et al., 2017). The phagophore

elongates and encircles the cytosolic substances to form double membrane-bound

autophagosomes that subsequently fuse with the vacuole to release the internal vesicle for

degradation (Liu and Bassham, 2012) (Figure 3).

Figure 3: Schematic Diagram of Macroautophagy.

Source: Tong et al., (2020).

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2.3.2 Microautophagy

Microautophagy is a non-selective lysosomal degradation process. In micro-autophagy,

cytosolic components are directly taken up by the lysosome itself through invagination or

deformation of the lysosomal membrane and it does not require autophagosome as transport

intermediary. During microautophagy, cytosolic proteins or entire organelles are congregated

together near the vacuole and become encapsulated by an invagination or protrusion of the

vacuolar membrane. This forms an intravacuolar vesicle called an autophagic body, which is

released to the vacuolar lumen by membrane scission and is degraded (Marshall and Vierstra,

2018). Microautophagy has a role in maintaining homeostasis and cell survival (Li et al,

2012). On the types of autophagy known, microautophagy has not been investigated

extensively yet it also serves as an important tool for biosynthetic transport, metabolic

adaption, organelle remodeling and quality control.

2.3.3 Chaperon Mediated Autophagy

Chaperon Mediated Autophagy (CMA) has only been described in mammalian cells and has

two unique properties which sets it apart from the other forms of autophagy; the mechanism

of delivery of substrate proteins to lysosomes and it is highly specific in action. Targeted

proteins are translocated across the lysosomal membrane in a complex with chaperone

proteins (Massey et al., 2004). In CMA, proteins bearing a particular pentapeptide motif

(KFERQ) are selectively degraded through direct translocation into the lysosome by Hsc 70.

The major role of this pathway in cellular metabolism was predicted to be the supply of free

amino acids generated following protein degradation. However, recent studies have shown

that impaired CMA significantly alters glucose and lipid metabolism and, consequently, the

10
energy metabolism of the whole organism, while also playing an important role in the

regulation of cellular metabolism in response to various nutrients (Tasset and Cuervo, 2016).

2.4 Selective Autophagy

Non-specific autophagy induced in response to nutrient or energy deprivation enables cell

survival. Macroautophagy can also be highly specific, and in this mode functions more in cell

maintenance and homeostasis (Chen and Klionsky, 2011; Isakson et al., 2013.). Specific

autophagic cargoes can include, but are not limited to peroxisomes, mitochondria, and

ubiquitinated proteins (Lee et al., 2012; Till et al., 2012; Weidberg et al., 2011).

Pexophagy (selective degradation of peroxomes) is important for a majority of the turnover

of peroxisomes under normal growth conditions (Huybrechts et al., 2009). Peroxisomes can

be degraded under starvation conditions, during which they can be specifically recognized by

autophagosomes through binding of LC3-II to PEX14, a component of the peroxisomal

translocon complex found on the peroxisomal membrane (Hara-Kuge and Fujiki, 2008).

Peroxisomes aid in varieties of metabolic functions and the negative effects of peroxisomal

dysfunction on human health (Till et al., 2012).

Mitophagy involves the selective degradation of mitochondria which is important in

mammals for steady-state turnover of these organelles (Tal et al., 2007) and for the

development of certain cell types and the clearance of damaged mitochondria (Kim et al.,

2007; Kundu et al., 2008; Schweers et al., 2007). In order for mammalian red blood cells to

mature, mitophagy is used to remove mitochondria from the immature cells (Kundu et al.,

2008; Mortensen et al., 2010; Zhang et al., 2009). The clearance of damaged mitochondria

proceeds in a way that the cytosolic E3 ubiquitin ligase PARK2/Parkin is recruited to

damaged mitochondria by the mitochondrial outer membrane kinase PINK1 (PTEN Induced

Kinase 1). PARK2 ubiquitinates mitochondrial substrates, leading to mitophagy (Youle and

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Narendra, 2011). In healthy mitochondria, PINK1 is imported into the mitochondrial inner

membrane, and subsequent cleavage by the mitochondrial processing peptidase (PMPCB)

and presenilin associated, rhomboid-like protease (PARL) leads to its eventual degradation

preventing accumulation of PINK1 on the mitochondrial outer membrane, thereby, avoiding

mitophagy of healthy mitochondria (Jin et al., 2010; Meissner et al., 2011.). The genes

encoding both PINK1 and PARK2 are mutated in autosomal recessive Parkinson disease

(Valente et al., 2004), emphasizing the importance of mitophagic clearance of damaged

mitochondria in maintaining cellular health.

Xenophagy is the degradation of intracellular pathogens. The ubiquitin-binding protein

SQSTM1/p62 targets intracellular bacteria for degradation through xenophagy (Zheng et al.,

2009). SQSTM1 is also important for the clearance of ubiquitinated protein aggregates by

acting as an adaptor protein that interacts with LC3-II to target aggregates for specific

degradation in a process termed aggrephagy (Øverbye et al., 2007).

Other forms of selective autophagy include; chlorophagy (degradation of chloroplasts),

reticulophagy (degradation of endoplasmic reticulum), proteaphagy (degradation of

proteasomes), ribophagy (degradation of ribosomes), degradation of the pre-autophagosomal

structure, degradation of TSPO (tryptophan-rich sensory protein), and the degradation of

brassinosteroid-responsive transcription factor BES1 (Masclaux-Daubresse et al., 2017;

Yoshimoto and Ohsumi, 2018).

2.5 Interplay of Autophagy and Reactive Oxygen Species (ROS)

Reactive oxygen species signifies a category of active-oxygen containing compounds

generated from aerobic metabolism (Siti et al., 2015). ROS are produced in cells through

metabolism of oxygen and, when at high levels, can function as destructive molecules,

actively participating in microbial elimination within phagocytes and also contributing to

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genomic instability, thereby resulting in cell death and/or tumorigenesis. In more recent

years, however, ROS at lower physiological levels have been recognized as intracellular

signal transduction molecules that regulate kinase-driven pathways, in turn mediating cellular

responses to external stimuli or challenges, such as growth factors, nutrient deprivation, or

hypoxia (Gough and Cotter, 2011). Oxidative stress is associated with elevated intracellular

reactive oxygen species (ROS). Intracellular ROS are mainly (approximately 90%) generated

by the electron transport chain in the inner membrane of mitochondria and consist of

hydrogen perxoide (H2O2), superoxide anion (O2-) and free radicals i.e. hydroxyl radicals

(OH·) (Shadel and Horvath, 2015; Murphy, 2009). ROS can oxidize organelles, nucleic acids,

proteins and lipids, which results in cellular damage (Pizzino et al., 2017).

Amino acid deprivation induces the formation of H 2O2 in mitochondria in a Class III PI3K-

dependent manner, and this ROS is essential for the induction of autophagy in response to

starvation (Scherz-Shouval et al., 2007). Specifically, the Cys81 residue near the catalytic site

of Atg4 is a direct oxidation target by H2O2; its oxidized form inactivates the protease activity

of Atg4 and prevents the delipidation of LC3 without affecting the C- terminal processing of

LC3 by Atg4, thus leading to increased autophagosome formation. In a reducing

environment, the Atg4 protease remains active and delipidates LC3, thereby suppressing

autophagosome membrane formation and resulting in autophagy inhibition. ROS triggers the

autophagy pathway to maintain redox homeostasis and remove oxidized organelles with other

components (Liu et al., 2020).

The autophagic pathway can also modulate the cellular levels of ROS through mitophagy.

Mitophagy is essential for the turnover of normal mitochondria, as well as the removal of

damaged mitochondria, which are primary sources of intracellular ROS. (Lemasters, 2005;

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Youle and Narendra, 2011). Autophagy deficient cells have significantly higher ROS levels

compared to their wild-type counterparts (Mathew et al., 2009; Kongara et al., 2010).

On the other hand, ROS can also modulate autophagy indirectly by affecting the activity of

the autophagy regulator mTORC1. The GTPases Rheb and Rag regulate the activity and the

spatial localization of mTORC1, respectively, in response to amino acid and growth factor

availability. Rheb is in turn regulated by the GTP activating proteins (GAP) TSC1/TSC2:

upon amino acid and growth factor stimulation, PKB/Akt inactivates TSC2 by

phosphorylation, resulting in Rheb and mTORC1 activation and concomitant suppression of

autophagy (Zoncu et al., 2011). An earlier study indicated that mTORC1 activity increases in

the presence of oxidizing agents, such as diamide or the cysteine oxidant phenylarsine oxide

(PAO) (Sarbassov and Sabatini, 2005) and more recent work demonstrated that PAO

activates mTORC1 via Rheb (Yoshida et al., 2011). ROS-induced inactivation of PTEN

(phosphatase and tensin homolog) can result in increased mTORC1 activity via the PI3K–Akt

pathway, thus leading to suppression of autophagy. These studies indicate that ROS may

negatively regulate autophagy by mTORC1 induction via oxidation and inactivation of PTEN

and TSC1/2, and suggest that other mechanism(s) protective against mTORC1 activation

must be simultaneously active, so that autophagy is allowed to proceed as a prosurvival cell

function under oxidative stress. ROS can inhibit autophagy by directly oxidizing Atg proteins

(Atg7 and Atg10) or inactivating autophagy modulators (TFEB and PTEN) (Filomeni et al.,

2010; Cao et al., 2009; Su et al., 2018) (Transcription factor EB).

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 CHAPTER 3

AUTOPHAGY IN HUMAN HEALTH

Autophagy allows the body to break down and reuse old cell parts so that the cells can

operate more efficiently, it is therefore useful in maintaining cell growth and development.

Biological and physiological processes such as inflammation, apoptosis, cell proliferation and

metabolism influence the pathogenesis of human diseases and understanding of these

processes is necessary in the identification of diagnostic and therapeutic targets. Autophagy is

implicated widely in pathophysiological processes (cancer, metabolic and neurodegenerative

disorders) and in physiological responses to exercise and aging (Levine and Kroemer, 2008;

Mizushima et al., 2008; Ravikumar et al., 2010).

Autophagy takes direct part in the regulation of developmental programs (Mizushima &

Levine, 2010; Allen & Baehrecke, 2020), maintenance of stem cell self-renewal potential

(Chen et al, 2018; Dong et al, 2021), cellular differentiation and plasticity (Boya et al, 2018;

Clarke & Simon, 2019). The repercussions of autophagy on organismal homeostasis have

spurred considerable efforts toward the identification of clinically actionable targets to

modulate the autophagic pathway to prevent or treat diseases, in multiple pathological

circumstances (Galluzzi et al, 2017). Homeostasis is maintained within organisms through

the physiological recycling process of autophagy and has shown a great importance in the

regulation of fundamental functions in the cell to varied diseases.

3.1 Physical factors that can induce autophagy

Autophagy is induced when cells are stressed and then go into survival modes. The following

factors have been shown to induce autophagy

1. Fasting/Calorie Restriction (CR): Fasting means to stop eating for a certain amount

of time. This process deprives the body of nutrients which forces the body to
15
 repurpose cellular components to function. CR means decreasing the number of

energy unit/calorie your body consumes. This forces cells into autophagy to

compensate for lost nutrients. CR helps to prolong lifespan and also support healthy

human aging (Chung et al., 2013). Under starvation, TOR complex is inactivated

leading to autophagy induction. Degraded molecules are then transported back into

the cytosol where they can be used to synthesize proteins or oxidized by mitochondria

to generate ATP for cell survival. (Chen and Kilonsky, 2011; Mehrpour et al., 2012).

2. Exercise: Physical exercise has several health benefits including life-span expansion

and prevention against various diseases (Handschin and Spielgelman., 2008) with the

benefits overlapping with protective functions of autophagy. During autophagy,

degraded materials are released back into cytoplasm for energy and protein

metabolism (Feng et al., 2014). Studies in animals show that autophagy is necessary

for enhanced endurance (Lira et al., 2013), mitochondrial biogenesis (Ju et al., 2016)

and tissue remodeling in skeletal muscle (Ulbricht et al., 2015).

3.2 Effect of autophagy on T cell function

All living organisms undergo continuous renovation in order to replace old cells with new

ones. Autophagy is an important process needed for cells to degrade and recycle intracellular

components so as to generate macromolecular precursors and produce energy. Autophagy

recycles damaged parts into fully functioning cell parts, gets rid of non-functional cells and

destroys pathogen in a cell that can damage it like viruses and bacteria. Several studies have

shown specific regulation of two different forms of autophagy (macroautophagy and CMA)

in peripheral T cells. Regarding specific signals that are involved in activation of

macroautophagy in T cells, initial studies reported that CD4+ an CD8+ T cells upregulated

macroautophagy in response to T cell receptor (TCR) engagement (Pua et al., 2007; Hubbard

et al., 2010). As in many other cell types, T cells can induce autophagy in response to

16
starvation (Li et al., 2006) and also in response to signaling that regulates T cell activation

(Pua et al., 2007; Botbol et al., 2015). As opposed to starvation-induced autophagy,

activation-induced autophagy appears to be independent of mTOR activity. In CMA, this

form is induced in response to TCR engagement (Valdor et al., 2014). CMA is a selective

form of autophagy that targets cytosolic proteins that present CMA-targeting motifs

biochemically related to the KFERQ pentapeptide (Dice, 1990). Activation of CMA after

TCR engagement responds to upregulation of the expression of LAMP-2A, mediated by the

increased generation of reactive oxygen species in activated cells (Valdor et al., 2014).

a. Cell Homeostasis: T cells lacking important Atg proteins suffer poor organelle

turnover (Jia and He, 2011; Jia et al., 2011). Mitophagy regulated mitochondrial

turnover is necessary for reduction of mitochondrial content when evolving from

thymocytes into mature peripheral naïve T cells. Atg-deficient T cells accumulate

mitochondria functionally altered, resulting in ROS accumulation which translates

into higher rate of cell death (Pua et al., 2009).

b. Cell Metabolism: Autophagy provides cells with substrates needed to synthesize

new cellular components and obtain energy as a response to starvation.

Macroautophagy regulates metabolic pathways to modulate cell function and

differentiation (Dowling and Macian., 2018). CD4+ T cells deficient in atg7 show

reduced production of ATP in response to TCR engagement, anaerobic glycolysis

and mitochondrial respiration is reduced when lysosomal activity is blocked

(Hubbard et al., 2010; Mocholi et al., 2018). CMA regulates carbohydrate and lipid

metabolism in liver, level of several enzymes and key upstream metabolic regulators

but modulation of T cell metabolism is yet to be known (Schneider et al., 2014;

Gomes et al., 2017).

17
3.3 Role of autophagy in healthy aging

Recent studies reported that autophagy is involved not only in disease but also in aging and

life span extension. Additionally, autophagy function has been demonstrated to decline with

age (Uddin et al., 2012). Aging process is marked by the molecular and cellular changes over

the years that results in the deterioration of physiological parameters important to keeping

organism alive and wealthy. At cellular level, aging is characterized by aggregation and

accumulation of misfolded proteins and disabled organelles in a progressive way that may

lead to cell homeostasis interruption, increasing risk of cell death (Lopez et al., 2013, Escobar

KA et al., 2019). Aging is associated with reduced autophagy potential and it has been shown

that autophagic inhibition may result in premature aging (Rubinsztein et al., 2011). With the

onset of aging, autophagy subsides, leading to the reduced formation of autophagic vacuoles

and improper fusion of the vacuoles with the lysosome which causes an impairment in the

protein flux with an accumulation of autophagic vacuoles in old tissues (Cheng et al., 2005;

Cuervo et al., 2005). Reports have documented that signaling pathways play a pivotal role in

controlling longevity. The most studied pathway is the IGF-1 (Insulin-like growth factor 1)

pathway (Cheng et al., 2005; Cuervo et al., 2005). A disruption of this pathway has been

found to extend longevity in different groups of species via mitigation of stress responses

(Cheng et al., 2005). The pathway includes PtdIns 3-kinase, tyrosine kinase receptor and

Akt/PKB (Protein kinase B). Akt/PKB has been discovered to be a potent positive regulator

on mTOR (autophagic inhibitor). Therefore, regulation of the pathway induces autophagy

confirming the link of IGF-1 pathway to autophagy, which is extensively connected to the

aging process.

Autophagy is upregulated during CR (Bagherniya et al., 2018) which leads to extension of

lifespan across phyla (Taormina and Mirisoia, 2014) while also reducing incidence of age-

related disorders (Bordone and Guarante 2005; Escobar et al., 2019). CR studies in non-

18
human primates have shown lifespan improvement and delayed onset of age-related

disorders. According to Mizushima et al. (2004), organisms can survive periods of starvation

by differentially inducing autophagy in an organ-specific manner in the liver, pancreas,

kidney and muscle while sparing the brain.

3.4 Autophagy in neurodegenerative diseases

The autophagy machinery is conserved across species, but because the brain possesses

precise mechanisms regulating its nutrient and energetic supply, basal autophagic flux was a

relatively late discovery in healthy neurons (Boland & Nixon, 2006). The first demonstration

of the relevance of autophagy in brain were findings in the Atg5, Atg7 knockout mice (Hara

et al., 2006; Komatsu et al., 2006).

Neurodegenerative diseases are characterized by the progressive loss of functions of neurons

in the brain. This loss of function can lead to a decline in cognitive abilities and eventually

death. Age is a major risk factor for all neurodegenerative diseases such as Alzheimer’s

disease, Parkinson disease, polyglutamine (polyQ) expansion diseases such as Huntington’s

disease etc. (Niccoli and Partridge, 2012). Common features in their parthenogenesis are

mitochondrial dysfunction and the accumulation of protein aggregates as a result of mutation

and impaired clearance mechanism (Jellinger, 2010; Goerdert et al., 2010.).

Activation of autophagy plays an important role in neurodegenerative therapeutics providing

an enthralling platform in the field of medical science (Abdullah et al., 2015). The age-

dependent functional decline of autophagy and the consequential attenuation of proteostasis

and accrual of proteotoxicity over time is thought to have contribution to the

development/progression of diseases. Neurons are post-mitotic cells unable to undergo

cytokinesis, since they cannot segregate the proteotoxic damage from daughter cells upon

mitosis, post-mitotic neurons are more susceptible to age-associated proteotoxicity. Beyond

19
clearance of misfolded proteins, autophagy likely plays important role in the clearance of

aggregate-prone mutant protein associated with several different neurodegenerative diseases.

1. Alzheimer’s disease

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder and the most common

cause of human dementia accounting for approximately 60%-80% of cases (Crous-Bou et al.,

2017). It is characterized by the accumulation of two proteins, namely Tau tangles/

intracellular MAPT and extracellular amyloid β-peptide which progressively leads to

neuronal cell death and decline in cognitive functions (Blennow et al., 2006). It is believed

that AD is the consequence of multiple risk factors including age, family history, genetic

background and brain injury. Relation of autophagy to AD originates from the observation of

expansion of autophagic components in AD brains (Nixon et al., 2005).

Recent studies show that mitophagy defects are closely related with AD development (Wang

et al., 2019). Mitophagy is an autophagic process of selectively removing excess or damaged

mitochondria (Lemasters, 2005). According to age of onset, AD is categorized into early-

onset AD (EOAD < 65 years) and late-onset AD (LOAD > 65 years). Many EOAD’s are

caused by the pathogenic mutation in three genes; APP, presenilin 1 and 2 (PSEN1 and

PSEN2) and is inherited in an autosomal dominant manner (Hardy, 2017; Tang and Gershon,

2003). Aβ peptide results from the degradation of Amyloid precursor protein (APP). APP, a

transmembrane glycoprotein (type I) is the key molecular driver of AD pathogenesis

(O’Brien and Wong, 2011). APP is ubiquitously present in the brain and is involved in the

building of synaptic network as well as regulating neurogenesis (Zhang et al., 2011).

Accumulation of amyloid-β peptide results in impaired fusion of autophagosomes with the

lysosomes. Proper formation and degradation of autophagosome is critical for normal

autophagic flux. In healthy neurons, low basal activity was detected because of the quick

20
subsequent degradation of autophagosome by lysosome. In hippocampus neurons of AD

mice, abnormal accumulation of immature autophagic vacuoles (AVs) in axon was observed

before synaptic and neuronal loss (Sanchez et al., 2012). CMA has been shown to play

important role in both Tau tangle formation and Aβ peptides generation and its activity is

impaired in aged cells (Koga and Cuervo, 2011).

Autophagy facilitates degradation and clearance of APP (Zhou et al., 2011) as well as all

APP cleavage products including Aβ (Sun et al., 2016; Son et al., 2012) and APP-CTFs (C-

terminal fragments). Accumulation of immature AVs in AD brains and in APP/PS1

transgenic mice may present a novel source for Aβ generation (Nixon et al., 2005).

Autophagy is also involved in the secretion of Aβ. Studies show that deficiency in autophagy

results in deficiency of Aβ secretion (Nilsson and Saido, 2014). On the other side, Aβ

peptides could also regulate autophagy. Aβ40 could induce autophagy in endothelial cells and

impair neurovascular regeneration (Hayashi et al., 2009). Emerging advances regarding

autophagy have indicated its role as protective factor in the early phase of AD but can also

help in the progression of AD in the late phase.

2. Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder

characterized clinically by motor system dysfunction and neuropathological by progressive

loss of dopaminergic neurons. A distinguishing factor of PD is the presence of round,

intracytoplasmic bodies called Lewy bodies present in the nucleus of neurons. These bodies

consist of an insoluble aggregated protein called α-synuclein which is susceptible to

degradation through CMA (Cai et al., 2016; Kesidou et al., 2013). PD has been examined to

contain mutations in certain genes; α-synuclein (SNCA), glucocerebroside (GBA), Parkin

PBR E3 ubiquitin protein ligase (PRKN/PARK2), PTEN-induced putative kinase 1 (PINK1)

21
and leucine-rich repeat kinase 2 (LRRK2) responsible for the early occurrence of the disease

(Alcalay et al., 2010). α-synuclein encoded by SNCA gene, is a presynaptic protein in

neurons of the central nervous system.

Recent studies show that selective autophagy clears neuron released SNCA through the

autophagy receptor SQSTM1/p62 in microglia offering protection of dopaminergic neurons

(Choi et al., 2020). Activation of autophagy decreases accumulation of SNCA and

alternatively, uncontrolled expression of wild-type or mutated variants of SNCA reduces

autophagic flux or disturbs TFEB-mediated lysosomal biogenesis by preventing nuclear

translocation of TFEB (Nakamura et al., 2019; Decressac et al., 2013). Overexpression of

wild-type α-synuclein compromise autophagy in mammalian cell lines and transgenic mice

by inhibiting RAB1A (GTPase involved in the early secretory pathway) and results in

mislocalization of atg9 and decreased formation of omegasomes an autophagic structure

associated with the endoplasmic reticulum (ER) (Winslow et al., 2010; Axe et al., 2008).

3. Huntington’s Disease

Polyglutamine related diseases are dominant late onset genetic disorders that are manifested

by progressive neurodegeneration, leading to behavioral and physical impairments. The role

of autophagy in disorders caused by polyglutamine (polyQ) expansion has been highlighted

in extensive experiments (Jimenez-Sanchez et al., 2012). Huntington’s disease is marked by

the CAG trinucleotide repeat that is expanded in the gene coding for the huntingtin (HTT)

protein. polyQ expansion in HTT is the pathogenic driver of HD (Zheng et al., 2010) and its

gravity is a direct function of polyQ length. The length of the polyQ tract is crucial, because

longer tracts are more prone to form aggregates. These mutant proteins accumulate and form

intracellular inclusions, and previous studies showed that autophagy degrades not only these

inclusions but also soluble mutant proteins. There is a contrast in the function of wild-type

22
and mutated HTT in the regulation of autophagy (Martin et al., 2015; Ashkenazi et al., 2017).

Wild type HTT participates in regulation of basal autophagy due to its role in selection of

autophagic cargo (Ochaba et al., 2014; Rui et al., 2015). Expression of mutant HTT;

a. Negatively affect autophagosomal cargo recognition through dysregulated interaction

with SQSTM1/p62 (Martinez-Vicente et al., 2010; Rui et al., 2015).

b. Disrupts ability of wild-type HTT to bind ULK1 and release it from negative

regulation of MTOR in order to activate autophagy (Rui et al., 2015).

c. Interferes with the regulatory interaction between ATXN3 (ataxin 3) and BECN1

compromising the response of neurons to starvation (Ashkenazi et al., 2017).

d. Disturbs axonal autophagosome transport (Wong and Holzbur, 2014).

Overexpression of wild-type HTT restores autophagy and clears mutated HTT (Zheng et al.,

2010). Defective autophagy caused by heterozygous depletion of autophagy scaffold

accelerates age of onset and progression of HD (Fox et al., 2020)

3.5 Autophagy in cancer

Autophagy has also been studied in cancer and it has been considered to be a tumor-

suppressive mechanism during tumor initiation and malignant transformation. Autophagy

helps in the removal of damaged cells and organelles resulting in limited cell proliferation

and genomic instability. The link between autophagy and tumorigenesis is through a tumor

suppressor p53 gene, a mutation that facilitate the growth of cancer. Earlier works shows that

an inverse relationship exists between the autophagic process and the p53 gene which in turn

regulates cancer progression (Cordani et al., 2017).

The p53 gene counteracts autophagic process through Akt/mTOR. Beclin 1 is another

autophagy gene reported to be a tumor-suppressor gene. Studies show that the Beclin 1 locus

is deleted in up to 75% ovarian cancer and up to 50-70% in breast cancer (Liang et al., 1999).

23
Reports have shown that epidermal growth factor receptor (EGFR) inhibits autophagy by

binding to Beclin 1, thereby, allowing cancer cells to survive against stress conditions.

Evidence shows that autophagy can also play a protective role in the progression of cancer.

Highly burgeoning cancer cells require cellular building blocks for their metabolism and

energy production. During this stage, autophagy helps to provide essential cellular

intermediates necessary for the cancer progression. Tongue squamous cell carcinoma (TSCC)

shows cisplatin resistance via autophagy activation. Treatment of TSSC with chloroquine

(CQ) and Beclin1 siRNA increased cisplatin sensitivity, strengthening the fact that autophagy

inhibition can be a potential target for treating TSCC (Liao et al., 2018). Oral Squamous Cell

Carcinoma (OSCC) shows resistance against cisplatin which is regulated by increased

autophagic flux. The Fadu-CDDP-R (Fadu cisplatin resistant) cells showed increased

autophagic markers (such as Beclin1, Ulk1, Atg5, Atg7 and Atg14), surface resistant marker

CD44 was found to be decreased in Atg14-deficient Fadu cells (Naik et al., 2018).

Autophagy plays prominent role in cancer progression by favoring cellular metabolites and

redox homeostasis. In hypoxic conditions, cancer cells consume glucose through anaerobic

glycolysis (Warburg effect) to provide unlimited access to glycolytic intermediates. In

addition, cancer cells exhibit higher glutamine utilization leading to autophagy induction

through mTOR inactivation for survival of the cancer cells in harsh microenvironmental

conditions (Victor et al., 2015). Figure 4 shows the interaction of autophagy with various

diseases and disorders.

24
Figure 4: Role of autophagy in various diseases and disorders.

Source: Source: Saha et al., (2018)

3.6 Autophagy in Cellular Death

When cells are no longer needed, they commit suicide by activating an intracellular death

program. This process functions as a component of homeostasis, approximately one million

cells are lost per minute throughout life in the hematopoietic system (Levine and Ucker,

2019). Forms of cell death is divided into three types (Edinger and Thompson., 2004).

Type I is apoptosis which is characterized by the condensation of cytoplasm and chromatin,

DNA and cell fragmentation into apoptotic bodies followed by removal and degradation of

the damaged cells by phagocytosis. Cells undergo apoptosis during development to regulate

tissue/organ shape and function (Saikumar et al., 1999). The primary executioners of

25
apoptosis are a family of enzymes known as caspases (CASPs). They cleave protein between

the cysteine and aspartic residues (Cohen, 1997; Galluzzi et al., 2018).

Type II is autophagy which is an evolutionary process allowing cells to survive starvation and

other stress by promoting sequestration and degradation of bulk cytoplasm, damaged

organelles and protein aggregates (Hughes and Rusten, 2007; Ohsumi, 2014; Wanderoy et

al., 2020).

Apoptosis and autophagy are both important processes used to maintain homeostasis.

Apoptosis maintains this role via dismantling damaged or unwanted cells while autophagy

recycles selective intracellular organelles and molecules. The two processes are regulated by

common factors and share common cmponents. Pro-apoptotic signals (e.g. those transduced

by BH3 only proteins) induce autophagy, alternatively signals that inhibit apoptosis (e.g.

through Bcl-2 family members) also inhibit autophagy. Atg5 can undergo calpain-mediated

cleavage to generate a pro-apoptotic fragment that functions in the intrinsic mitochondrial

death pathway. (Yousefi et al., 2006).

Type III is necrosis which is uncontrolled death of a cell following an injury, resulting in

spillage of the contents of the cell into surrounding tissues and inflammation. Other cellular

death includes; ferroptosis, pyroptosis, necroptosis, parthanatos, entosis, MPT-driven

necrosis (mitochondrial permeability transition), NETotic cell death, oxytosis and mitotic

catastrophe (Galluzzi et al., 2018; Tang et al., 2019; Nirmala and Lopus, 2020)

26
 CHAPTER 4

AUTOPHAGY IN PLANTS

Autophagy is a process conserved throughout eukaryotes for degradation and recycling of

cytoplasmic components during development or environmental stress. Three major types of

autophagy have been discovered in plants, microautophagy, macroautophagy and mega-

autophagy (Fig. 5) (Avin-Wittenberg et al., 2018). During microautophagy, cytoplasmic

components are taken up by the vacuole through the invagination of the tonoplast (Nakamura

et al., 2018). When macroautophagy is activated, the phagophore forms around cargo,

expands and finally seals as a double-membrane vesicle called an autophagosome with the

outer membrane fusing with the tonoplast and releasing an autophagic body into the vacuole

for degradation (Marshall and Vierstra, 2018). In mega-autophagy, the tonoplast membrane

ruptures to release the vacuolar hydrolases directly into the cytoplasm, where it degrades

cytoplasmic materials (Hatsugai et al., 2004; Nakatogawa, 2020). Mega-autophagy involves

massive degradation of cells. The rupture of tonoplast leads to the release of large amounts of

hydrolases into the cytoplasm and the cytoplasm with the cell wall become degraded leading

to cell death (Van Doorn and Papin, 2014; Marshall and Vierstra, 2018). Microautophagy

decreases tonoplast membrane area and macro-autophagy provides new lipid material to the

tonoplast. More than 40 atg genes have been described in Arabidopsis thaliana

(Arabiodopsis) (Chung, 2019). In plant development, autophagy helps in basal cell function,

housekeeping duties and response/ tolerance to biotic and abiotic stress (Rodriguez et al.,

2020).

27
Figure 5: Types of autophagy found in plants.

Source: Avin-Wittenberg et al., (2018); Marshall and Vierstra, (2018).

4.1 Autophagy in Vegetative Growth

4.1.1 Seed Development

Life cycle of flowering plants begin with a seed. Atg mutations causes plants to produce

fewer seeds. In Arabidopsis, several atg mutations show decreased seed production and some

atg genes are up-regulated during seed maturation (Liu and Bassham, 2012; Angelovici et al.,

2009). Role of autophagy in seed development has not been explained at mechanism level but

studies show that autophagy may contribute to transport of seed storage proteins and seed

germination. (Berardino et al., 2018; Honig et al., 2012). Some research also show that

autophagy is involved in plant oil production, oil accumulates in embryonic tissues and

endosperm during seed development and germination (Ortiz et al., 2020). Overexpression of

atg5 or atg7 increases both seed yield and fatty acid content as seen in Arabidopsis (Minina

et al., 2018).

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4.1.2 Root Development

Structurally, plant roots are divided into three zones; meristematic, elongation and maturation

zone, while using cross section, the roots are divided into three levels; dermal, cortex and

vascular tissues (Evert, 2007; Wojciechowska et al., 2021). During root development,

autophagy helps in its establishment and functional differentiation. Atg8 genes are located in

the root caps and maturation zone which corresponds to relevant protein degradation

(Sláviková et al., 2005). In Arabidopsis roots, autophagy is required for ground tissue

differentiation in order to degrade cytoplasmic material and for vacuole formation from the

meristem to the elongation zone (Inoue et al., 2006). Also, atg8f protein localizes to

autophagy-like structures in the central vacuole, suggesting that autophagy also determines

vacuolar generation in cortical parenchymal cells (Sláviková et al., 2005).

In addition to root development, autophagy plays vital role in root senescence

(Wojciechowska et al., 2018). During the first stage of senescence, autophagy transits cell

death, maintains cellular homeostasis and is also involved in the remobilization process, a

key step in senescence process (Wojciechowska et al., 2021).

4.2 Role of Autophagy in Leaf Senescence

Senescence represents the final stage of leaf development. It is characterized by the transition

from nutrient assimilation to nutrient remobilization (Masclaux et al., 2000). During

senescence, cells do not simply deteriorate and collapse, they undergo orderly changes that

modify their structures, metabolism and sink–source relationships (Thomas, 2013). Leaf

senescence occurs in a coordinated manner starting from the tip and margins toward the base

of the leaf in many plant species like Arabidopsis, wheat, barley, and maize. Plants are non-

mobile and cannot escape from biotic and abiotic stress conditions during development,

plants senescence the leaves to overcome this stress when they remobilize phloem-mobile

29
nutrients and energy gotten from leaf senescence is used to generate energy for developing

tissues and storage enzymes (Avila-Ospina et al., 2014). The involvement of autophagy in

senescence is supported by the observation that many Atg transcripts are upregulated in older

leaves. For example, in Arabidopsis, 15 Atg genes are upregulated during senescence (Breeze

et al., 2011).

Accumulation of lapidated Atg8 represents higher level of autophagy activity in the

yellowing area of a senescing leaf compared to the green area of the same leaf in maize

(Chung et al., 2009). Nitrogen is the main element remobilized during senescence (Havé et

al., 2017). Accumulation of nitrogen in seeds is lower in autophagy defective mutants

compared to wild type (Guiboileau et al., 2012). This shows that autophagy is necessary for

efficient nitrogen remobilization during leaf senescence and seed set.

4.3 Role of Autophagy in Nutrient Availability

4.3.1 Macronutrients

Two major nutrients required by plants for cellular function, plant growth and development

are carbon and nitrogen (Zheng, 2014). Sucrose and glucose, products of photosynthesis

serve as energy sources and also provides carbon skeleton for ammonium assimilation during

amino acid biosynthesis. Organic nitrogen nutrients such as amino acids and their resulting

proteins are the major building blocks of the cell.

In Arabidopsis, sucrose is the immediate product of carbon fixation which helps in plant

growth. Stored form is starch, it accumulates during the day time and during night time,

starch is degraded to sucrose for use. This process occurs in different species of plants

(Chung et al., 2009). Under insufficient light condition, not enough carbon can be fixed,

autophagy is one mechanism that enables the decomposition of carbon sources for energy

production. In Arabidopsis, autophagy facilitates transitory leaf starch degradation (Wang et

30
al., 2013). At seedling stage, autophagy contributes to cellular metabolism under carbon

starvation. Metabolic profiling of Atg mutant etiolated seedlings grown with no exogenous

sucrose revealed reduced levels of free amino acids in the mutants, while proteomic analysis

showed an accumulation of proteins (Avin-Wittenberg et al., 2015). An accumulation of total

proteins was also observed in mature Arabidopsis plants exposed to 3, 6 and 9 days of

extended darkness. However, an increase in total free amino acids was observed and

increased levels of tricarboxylic acid (TCA) cycle intermediates (Barrows et al., 2017).

Chlorophagy (selective chloroplast degradation) is involved in chloroplast photo-damage.

Under high light or UV radiation, accumulation of ROS damage the chloroplast envelope,

resulting in chloroplast degradation via selective microautophagy (Izumi et al., 2017; Izumi

et al., 2019). As the chloroplast is also the major nitrogen resource in plant cells, it plays an

important role in response to nitrogen starvation. Piecemeal degradation of chloroplast has

been characterized as a response to carbon starvation. However, the most well-characterized

type of chlorophagy is that of Rubisco-containing bodies (RCBs), composed of chloroplast

stroma. RCBs have been observed during carbon starvation after darkening individual leaves

(Ishida et al., 2008). Formation of RCBs is specific to the carbon status and not nitrogen

status of the plant, even though the breakdown of Rubisco also supplies nitrogen. It has been

postulated that partial decomposition of the chloroplast using RCBs may allow for

maintaining basic chloroplast functions during energy limitation without loss of complete

chloroplasts. Two roles of RCBs may exist; provision of a temporary energy source under

plant carbon restriction and efficient breakdown of chloroplasts under leaf carbon restriction

(Izumi et al., 2010).

Nitrogen availability in soil tends to be low which is why large amounts of nitrogen fertilizers

are used in agriculture (Good et al., 2004; Forde, 2002; Maathius, 2009). Nitrogen is utilized

in several stages of a plants life cycle. Inorganic nitrogen is taken from soil and is assimilated

31
into amino acid to serve as the primary source of nitrogen while nitrogen gotten from

remobilization of nitrogen from leaf proteins serves as major source of nitrogen (Li et al.,

2015; Masclaux-Daubresse et al., 2010). Nitrogen is remobilized in an organic form as amino

acids, small peptides, urea and inorganic nitrogen such as ammonium and nitrate. Amino

acids and peptides are assumed to be the predominant forms of remobilized nitrogen, linking

nitrogen remobilization to carbon remobilization (Havé et al., 2016). Chloroplasts are the

primary source for nitrogen recycling and remobilization from vegetative plant tissue.

Autophagy possibly has diverse roles during limited nitrogen conditions. Enhanced

autophagy in apple plants was linked to increased transcript levels of high-affinity nitrate

transporters and nitrate reductase, therefore positively regulating nitrate uptake and

assimilation during nitrogen starvation. The same study found increased transcript levels of

anthocyanin-related genes and an accumulation of anthocyanins in response to nitrogen

depletion in roots of apple and Arabidopsis seedlings with enhanced autophagy (Sun et al.,

2018).

Recent studies have demonstrated that lipid turnover and lipid-related pathways are strongly

affected in different atg mutants in Arabidopsis and maize under both nitrogen-sufficient and

nitrogen-deficient conditions (Barros et al., 2020; Fan et al., 2019). Lower levels of lipids in

nitrogen-deprived atg mutants might be explained by the upregulation of transcript levels of

lipases, as found in maize Atg12 leaves (McLoughlin et al., 2018). Moreover, the amounts of

unsaturated and long-chain lipids were reduced under nitrogen starvation, suggesting that

autophagy also influences lipid composition, pointing to extensive membrane lipid

remodeling (Megume et al., 2022; Havé et al., 2019).

4.3.2 Micronutrients

32
Phosphorus is an essential component of many biomolecules such as phospholipids,

nucleotides and ATP. Phosphorus starvation was shown to induce autophagy in different

plant species. In Arabidopsis roots, phosphorus limitation caused ER stress; due to low level

phosphorus, ER was degraded by autophagy (Liu et al, 2012; Naumann et al, 2019).

However, oversupply of nitrogen was seen to induce autophagy in Arabidopsis and

suppressed phosphorus starvation stress when phosphorus was deprived (Yoshitake et al.,

2020). Nitrogen oversupply disrupts the carbon/nitrogen ratio, mimicking fixed-carbon

starvation. Therefore, excess nitrogen under phosphorus depletion, just as fixed-carbon

starvation, induces the formation of RCBs.

In conditions of sulfur limitation, atg mutants are hypersensitive. The lower sulfur flux in atg

mutants reduced seed number and resulted in improper seed filling with sulfur and nitrogen,

emphasizing the role of autophagy in sulfur remobilization from the leaves to seeds (Lornac

et al., 2020). Chronic sulfur limitation induced transcript levels of several atg genes while

chronic nitrogen limitation had a much weaker effect on atg gene expression (Luo et al.,

2020). Sulfur remobilization may involve organic forms such as sulfur-containing amino

acids, glutathione, sulfur-containing secondary metabolites and inorganic forms like sulfate,

which accumulated in rosette leaves of the atg5 mutant. Under sulfur limitation, sulfur

remobilization efficiency in atg5 mutants was more affected than nitrogen remobilization

efficiency, indicating that mobile nitrogen poor sulfur containing molecules were missing in

the mutant compared with the wild-type (Lornac et al., 2020).

Metal micronutrients are essential to all forms of life. For instance, iron plays a significant

role in redox reactions during respiration in mitochondria and photosynthesis in chloroplasts

(Nouet et al., 2011). Copper is required for photosynthesis, respiration, ethylene perception,

ROS metabolism, and cell wall production in plants (Burkhead et al., 2009). Zinc is an

important catalytic or structural cofactor for many enzymes and transcriptional factors. Plants

33
must balance the uptake, utilization, and storage of these metals in order to maintain proper

ion homeostasis (Shinozaki et al., 2020; Shinozaki and Yoshimoto, 2021). Autophagic

induction has been shown to participate in the regulation of metal uptake. Transcript levels of

several atg genes and autophagic flux were induced by iron deficiency in Arabidopsis leading

to increased autophagic activity resulting in an increased turnover of a negative regulator of

iron absorption and improved plant growth under low iron conditions compared with atg

mutants (Zhang et al., 2021). When zinc is in excess, causing an iron deficiency, iron ions

can be remobilized from protein bound iron through selective autophagy. Although metals

are important for protein stability and cellular processes (Goldberg, 2003; Zhou et al., 2016),

high concentrations of metal ions can also have toxic effects. Heavy metals are known to be

strong inducers of oxidative stress due to the excessive accumulation of ROS that alters

cellular homeostasis. Excess accumulation of ROS can cause damage to a multitude of

cellular components, including proteins, lipids, chloroplast, and DNA (Hassan et al., 2017).

4.4 Autophagy in response to Abiotic Stress

Autophagy can be induced by various abiotic stresses including nutrient starvation, oxidative

stress, salt, drought and heat stress. When plants are exposed to these conditions, ROS

production acts a common signal to activate stress response which includes autophagy.

Nutrient starvation triggers autophagy and atg mutants exhibit premature senescence upon

C/N starvation. Using sucrose starvation in suspension-cultured cells, 30% to 50% of the total

protein is degraded over a two-day period (Takatsuka, 2004) and the decrease in total

proteins stems from non-selective degradation, rather than degradation of specific proteins

(Moriyasu and Ohsumi, 1996). Nutrient stress also affects TOR signaling, activating

autophagy production. Previous study shows that autophagy can balance zinc (Zn) and iron

34
(Fe) uptake in plants (Shinozaki and Yoshimoto, 2021). It increases zinc bioavailability in

plants when zinc levels in the environment are low (Shinozaki et al., 2020).

Heat stress can cause misfolding and denaturing of proteins, trigger ER degradation to

balance cellular homeostasis (Deng et al., 2011). Plants accumulate large amounts of

oxidized and insoluble proteins at unsuitable temperatures. These toxic proteins are

eliminated by inducing autophagy to improve plant resistance. Atg gene expression is up-

regulated in various plants, and more autophagosomes are accumulated under heat stress

(Zhai et al., 2016; Zhou et al., 2014). Heat stress induces expression of atg genes and the

accumulation of autophagosomes, autophagy is proposed to contribute to heat tolerance

(Yang et al., 2016; Zhou et al., 2014).

Involvement of autophagy in response to drought stress was first studied in Arabidopsis,

indicated by the upregulation of Atg18a and the induction of autophagosome formation by

osmotic stress (Liu et al., 2009). Drought stress increases expression of atg genes in crops

such as Atg2 in peppers (Zhai et al., 2016), Atg8a in millet (Li et al., 2016), Atg6 in barley

(Zeng et al., 2017), and Atg3 and Atg18a in apples (Wada et al., 2015; Wang et al., 2017).

High concentrations of salt (NaCl) leads to a reduced photosynthetic rate as well as excessive

energy consumption and accumulation of excess ROS (Yue et a., 2018). Autophagy helps in

regulating cellular homeostasis by activating pathway of plant salt tolerance.

4.5 Autophagy in response to Biotic Stress

Depending on nature of the pathogen infecting the plant, autophagy is shown to lead to

different outcomes (Leary et al., 2018). Autophagy is involved in plant immunity by

negatively regulating PCD (Liu et al., 2005).

Autophagy in plants during viral infection can either be anti-viral or facilitate progression of

the virus (Yang and Liu, 2022). It was discovered that autophagy defends DNA viruses from
35
infecting plants. The virulence factor βC1 from CLCuMuV is degraded by autophagy through

interaction with Atg8. βC1 acts as the first plant viral activator of autophagy to activate

autophagy by disrupting the interaction between Atg3 and glyceraldehyde-3-phosphate

dehydrogenase, a negative regulator of autophagy (Han et al., 2015). Autophagy also shows

antiviral effects during infection by positive-strand RNA viruses. For example,

overexpression of Beclin1/Atg6 inhibits TuMV viral RNA accumulation, while the knockout

of Beclin1 or Atg8a promotes its infection (Li et al., 2018). However, the molecular

mechanism of autophagy inhibiting TuMV infection is still to be investigated further. In

addition, autophagy also plays an antiviral role in negative-strand RNA virus infection. The

viral suppressors of the RNAi (VSR) protein P3 from rice stripe virus (RSV) can interact

with NbPI3P and can be degraded by autophagy, thereby inhibiting RSV infection (Jiang et

al., 2021). In this process, eukaryotic translation initiation factor 4A (eIF4A) acts as a

repressor by interacting with Atg5 to leave the Atg5–Atg12 interaction to inhibit autophagy

(Zhang et al., 2021). From these current studies, autophagy can inhibit viral movement, not

replication.

Autophagy can improve plant resistance to necrotrophic pathogens by limiting the hyper

sensitive response (HR) of host cells. Compared to the wild-type, multiple autophagy mutants

in Arabidopsis show reduced resistance to Botrytis cinerea, specifically, severe leaf

yellowing, larger lesion area, and more dead cells (Lai et al., 2011).

Bacteria have evolved in many ways to manipulate host cells for successful infection. Several

studies show that intracellular bacteria can manipulate autophagy as a pro- survival strategy

(Winchell et al., 2016). Pseudomonas syringae is a gram-negative bacterium having strong

aerobic and saprotrophic properties with its incidence of plant diseases ranking first in the top

10 bacterial plant diseases (Wang and Wang, 2017). Some studies show that autophagy has

reverse resistance to Pseudomonas syringae. Arabidopsis Atg5, Atg18a mutants significantly

36
reduce host cell HR after infection with the bacterium Pseudomonas syringae pathovar (pv)

tomato (Pst DC3000) with the effector proteins AvrRps4 or AvrRpm1 (Hofius et al., 2017).

Furthermore, when infected with Pseudomonas syringae, Arabidopsis atg5, atg10, and atg18a

mutant eaves do not spread cell necrosis, and the plants show obvious resistance (Lenz et al.,

2011).

CONCLUSION

Autophagy is self-eating and a natural regulatory mechanism for the destruction of damaged

cellular components and old functional proteins to facilitate intracellular recycling. It can be

induced by certain stimuli such as nutrient starvation, decreased ATP levels, decreased

insulin level as well as hypoxia. The process confines cytoplasm, improperly formed

proteins, long-lived proteins and organelles to the lysosomes for degradation.

Autophagy thus joins several bodily processes that helps to clear endogenous and exogenous

debris and hence facilitate and maintain homeostasis. As harmful as self-eating might sound,

it helps to get rid of toxic materials and repair them. It monitors growth, development and

differentiation of cells such as erythrocytes and endothelial cells as well as regulate cell

death, macromolecule and organelle turnover and oxidative stress. Whilst playing a

housekeeping role, the evolutional process retains and keeps beneficial substances vital to

proper cellular functions. It is involved in normal physiological, immunological, metabolic,

developmental and growth processes.

The impact of apoptosis (normal and controlled cellular death) and autophagy are interwoven

and are usually both implicated in a lot of health-related issues which include cancer,

abnormal protein accumulation and pathological cell stress processes. More in-depth research

into the roles of various types of autophagy in health, cancer and diseases (especially

37
neurodegenerative, genetic and even lifestyle related ones) could go a long way to maybe

creating alternative treatment regimens. Autophagy has also been implicated in re-emergence

of certain viral and bacterial infectious pathogens as well as antibiotics resistance that would

otherwise have helped to treat said infections.

38
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