Endoplasmic Reticulum in Healthy State

#### **Chapter 1**

## Navigating the Endoplasmic Reticulum: New Insights and Emerging Concepts

*Sikander Ali and Maria Najeeb*

#### **Abstract**

Endoplasmic reticulum (ER) is a membrane bound organelle adjacent to the nucleus in eukaryotic cells. It exists in the form of membranous sacs called "cisternae". It was first discovered by Emilio Veratti in 1902 and later named as 'Endoplasmic Reticulum' in 1953 after visualization through electron microscopy. There are two types of endoplasmic reticulum based on the presence of ribosomes i.e., 'rough' ER and 'smooth' ER. Rough ER is the site for protein synthesis and modification by glycosylation. While the smooth ER is involved in the metabolism of lipids and carbohydrates. Recently, it has been classified on the basis of membrane structure rather than appearance. It physically interconnects with the mitochondria and these sites are named as mitochondria-associated membranes (MAMs) that are crucial for Ca+2 homeostasis. Various mechanisms of ER signaling play vital role in physiology and the onset of disease. A thorough understanding of these mechanisms and their role in physiology and pathophysiology can be applied to develop new ER-targeted therapies.

**Keywords:** endoplasmic reticulum (ER), mitochondria-associated membranes (MAMs), Ca+2 homeostasis, ER-targeted therapies, perturbic ER functions

#### **1. Introduction**

Endoplasmic reticulum (ER) is a membrane bound subcellular organelle that appears as a network of tubules in the cytoplasm [1]. It was first observed in chicken fibroblast-like cells with the help of electron microscope. After 10 years, it was named by Porter in 1954 [2]. There are two types of ER based upon the presence or absence of ribosomes on its surface i.e., smooth endoplasmic reticulum SER and rough endoplasmic reticulum RER. Both of these could occur either interconnected or separately in compartments [3]. ER is a crucial site for various functions, including protein synthesis, storage and regulation of Ca2+ and lipid and glucose metabolism [3]. These range of functions indicate that ER plays important role in regulating metabolism and cell programming.

ER plays important role in protein synthesis and modification. After synthesis the proteins are translocated across the membrane of ER. Ribosomes attached to the RER membrane are involved in the synthesis of protein [4]. In addition, ER is also crucial

#### **Figure 1.**

*ER molecular machines and contact sites with other organelles. The ER forms multiple membrane contact sites with other organelles, including the endosomes and lysosomes (through STARD3, STARD3NL, Mdm1; panel 2), the mitochondria (through Mfn-2, Sig-1R, PERK; panel 3), and the PM (through ORAI1, STIM1, Sec22b, VAMP7; panel 4) with various functional implications.*

for Ca2+ signaling and homeostasis. A storage and transport system for Ca2+ comprising of Ca2+ channels, ATPase pumps, sensors and storage proteins. This chapter highlights a new aspect for ER classification, its functions, ER phagy and role of ER signaling in health and disease [5].

#### **2. Structure**

There are two types of ER based upon the presence or absence of ribosomes on its surface i.e., smooth endoplasmic reticulum SER and rough endoplasmic reticulum RER [2]. Both of these could occur either interconnected or separately in compartments. Recently, a new classification has been introduced based on its membrane structure instead of appearance. According to this, ER carry a nuclear envelope, cisternae and three way connected tubules. The ER is interconnected with many organelles in the cell and it is connected with mitochondria through specific sites, called mitochondria-associated membranes (MAMs), that are critical for maintaining Ca2+ homeostasis [6]. Its interaction with the plasma membrane is regulated by stromal interaction molecule 1 a protein-like structure and Calcium channel protein 1 [7]. Moreover, SEC22b a vesicle-trafficking also involved in the maintenance of this interaction [8]. ER interaction with endosomes is stabilized by lipid transfer protein 3, the protein involved in cholesterol maintenance in endosomes [9]. ER also play role in autophagy by interacting with endolysosomal system. These intracellular interactions are crucial for the functionality of the cell (**Figure 1**) [10].

*Navigating the Endoplasmic Reticulum: New Insights and Emerging Concepts DOI: http://dx.doi.org/10.5772/intechopen.105737*

#### **3. Functions**

ER is a crucial site for various functions, including protein synthesis, storage and regulation of Ca2+ and lipid and glucose metabolism. These range of functions indicate that ER plays important role in regulating metabolism and cell programming [3].

#### **3.1 Protein modification**

Secretory and transmembrane proteins are synthesized in ER. Their folding, maturation, quality control and degradation also occur in this organelle. As a result, only properly folded proteins are transported to their destination [4]. Approximately, 30% proteins are cotranslationally delivered to the ER, where chaperones are involved in their folding, packaging and post-translational modification [11]. Protein modification processes includes signal sequence cleavage, formation and breakage of disulfide bonds and lipid conjugation. Misfolded proteins are damaging to normal cellular functions and are tightly monitored. Protein misfolding is a regular process but aggravate during adverse conditions. In ER several regulatory systems ensure correction of misfolded proteins. Terminally misfolded secretory proteins are removed by ER associated degradation (ERAD) process [12]. Initially, proteins are encountered by an ER resident luminal and transmembrane protein machinery, then translocated into cytosol via channel called dislocon [13].

#### **3.2 Lipid synthesis**

ER is also crucial for synthesis of membrane constituents, lipid droplets fat accumulation as energy reservoir. Lipid synthesis takes place at membrane interfaces and organelle interaction sites. The ER membrane architecture dynamically altered according to cellular lipid concentration [14]. In order to maintain the cholesterol homeostasis in the body, ER carry a family of protein sensors. It also consists of various enzymes involved in synthesis of sterols and phospholipids (**Figure 1**) [15].

#### **3.3 ER export**

The proteins and lipids synthesized in the ER are delivered to their destined locations through secretory pathways. The export process is tightly controlled to maintain a steady anabolic flux because anomalies in secretion could lead to detrimental consequences to ER structure and functions [16]. Synthesis of ER COPII transport vesicles is crucial to this export process. Apart from COPII mediated transport several other mechanisms have been studied such as nonvesicular. For example, large lipoprotein cargo is transported out in another vesicle or stored in lipid droplets (**Figure 1**) [17].

#### **3.4 Ca2+ homeostasis**

Ca2+is a metallic ion plays key role as a secondary messenger in various intracellular and extracellular signaling events such as gene expression, translation, protein trafficking, and regulation of other cellular functions [6]. ER is the main reservoir of Ca2 **+** and important for its regulation. Myriad of cellular functions are regulated by Ca2+-dependent way in order to maintain the calcium level of entire cell. As a result, tight regulation of both ER and cytoplasmic Ca2+ concentration is essential to maintain enhanced intraluminal Ca2+ concentration redox potential as compared to the cytoplasm. A variety of mechanism employed by ER to maintain the concentration of Ca2+ inside and outside the membrane [5]. (a) ER membrane ATP-dependent Ca2+ pumps for cytosol-to-lumen transport; (b) ER luminal Ca2+-binding chaperones for sequestering free Ca2+ and (c) ER membrane channels for the regulated release of Ca2+ into the cytosol. These mechanisms are supported by a controlled interaction between the ER and other organelles, i.e., PM and the mitochondria.

#### **4. Perturbing ER functions**

Disturbance in ER function results in condition commonly known as 'ER stress'. In order to overcome ER stress and reestablish the homeostasis several adaptation mechanisms are activated inside the cell [18].

#### **4.1 Intrinsic ER perturbations**

In certain disease conditions such as cancer, diabetes and neurodegenerative diseases some cellular mechanisms lead to ER stress [19]. In case of cancer, rapid and uncontrolled cellular growth required high protein production that impact the ER system [20]. More specifically, in melanoma that has highest mutation rate the number of mutated proteins is increased that results in ER stress. In chronic myeloid leukemia, an oncoprotein is activated that promotes cell proliferation and disturbs Ca2+-dependent apoptotic response [21].

Many neurodegenerative diseases also disturb the ER homeostasis and lead to ER stress. For instance, motor neuron death is the consequence of mutations in the vesicle-associated membrane protein-associated protein B located in ER. It is mediated by the fluctuation of ER stress signaling [22, 23]. On the contrary, pancreatic beta cells involved in insulin production carry a complex and developed ER to control insulin production and use in response to high blood sugar level. Type 1 diabetes is associated with mutation induced ER stress, in this condition beta cells undergo apoptosis and insulin level reduced [24, 25]. Insulin mutation-related ER stress have also been observed in neonatal diabetes [26, 27].

#### **4.2 Extrinsic ER perturbations**

#### *4.2.1 Microenvironmental stress*

Microenvironmental ER stress occurs in tumorigenic cells. These cells rapidly proliferate that leads to deprivation of nutrients and oxygen in the microenvironment, resulting in local stress accompanied with hypoxia, starvation and acidosis, consequently ER stress, perturbation of protein and lipid biogenesis [28]. Nutrient scarcity, most importantly glucose distress facilitates ER stress by perturbing glycosylation.

#### *4.2.2 Exposure to ER stressors*

ER stressors are small molecules that stimulate ER stress mediated by a number of mechanisms [29]. These include molecules such as tunicamycin [30], or 2-deoxyglucose target the N-linked glycosylation of proteins. On the other hand, dithiothreitol prevents protein disulfide bond formation [31]. While Brefeldin A inhibits the ER to-Golgi trafficking, resulting in rapid and reversible inhibition of protein secretion [32].

#### *4.2.3 Exposure to enhancers of ER homeostasis*

Some molecules have been reported to stimulate and increase ER stress, such as peptides and proteostasis regulators. A most commonly used 4-phenylbutyric acid (4-PBA) prevents the aggregation of misfolded proteins in the ER [33]. In islet cells, to reduced ER stress a bile acid called Tauroursodeoxycholic acid (TUDCA) is present [34]. FDA has approved TUDCA as a drug for patients diagnosed with primary biliary cirrhosis [35]. The precise mechanism of action of these proteostasis regulators is still unknown.

#### *4.2.4 Temperature*

Mammals normal body temperature is 36–37°C necessary for viability and normal bodily functions. Fluctuations in normal body temperature could perturb cellular homeostasis consequently protein denaturation and aggregation [36]. In addition, an acute elevation in temperature, such as heat shock leads to fragmentation of both ER and Golgi [36]. In some mammalian cells and animal models mild elevation in temperature (up to 40°C) cause the development of thermotolerance, which is linked with increased expression of heat shock proteins and ER stressors [37, 38]. Moreover, mild hypothermia (28°C) stimulates mild ER stress in human pluripotent stem cells [39].

#### *4.2.5 Physiological ER stress signaling*

In physiological stress conditions, for instance increased secretory level or pathological stress, induced by aggregation of mutant protein could lead to imbalance between requirement for folded protein and ER potential to fold protein, consequently causing ER stress [40]. In response to stress, eukaryotic cells have developed signal transduction pathways, known as unfolded protein response (UPR) (**Figure 2**). These pathways are regulated by a group of proteins that sense ER stress. These proteins generate stress signals that protect cell from damage or induce cell apoptosis. A strong link between UPR signaling and human diseases has been reported [41].

The main objective of the UPR is to restore homeostasis and inhibit ER stress by employing the following mechanisms: (a) increasing protein folding ability through expression of protein-folding chaperones (b) Inhibition of protein translation by downregulating the ER protein load and facilitating the denaturation of misfolded proteins. But in case of acute stress the UPR stimulates programmed cell death [40]. UPR-mediated cell death is responsible for the onset of many diseases (**Table 1**), including cancer, type 2 diabetes, neurodegeneration, and atherosclerosis (40).

#### *4.2.6 Role of ER in metabolism*

The ER plays crucial role in the regulation of metabolic reactions. More specifically, the UPR pathway is involved in the regulation of glycolysis and it was recently reported that a regulatory protein mediates a metabolic decrease upon decrease in glucose level in neurons, suggesting an important role for the UPR as an adaptive response mechanism in relation to energy metabolism [42]. In addition, another signaling molecule known as mTOR maintains protein synthesis.

To maintain lipid content in the body ER plays an important role. Hepatocytes, liver cells carry SER in abundance, because along with protein synthesis, these cells also produce bile acids, cholesterol and phospholipids. Lipid accumulation leads to

#### **Figure 2.**

*A schematic of mammalian unfolded protein response (UPR) signaling. IRE1, PERK, and ATF-6 proteins reside at the ER membrane. In response to ER stress, they initiate a cascade of signal transduction outputs that control cell survival or death.*

lipotoxicity, which is the fundamental cause of metabolic diseases. ER carry various enzymes that are crucial for lipid metabolism. Cellular cholesterol level is regulated via signaling pathways. One of these pathways is SCAP/SREBP2, which converts cholesterol into oxysterols and eventually to bile acids. Similarly, level of intracellular fatty acid is controlled by ER by a bunch of enzymes including, desaturases, elongases, and beta oxidation cycles [43].

The UPR has also been reported important for amino acid metabolism. Amino acid synthesis is closely linked with demand for protein biogenesis during ER stress. In response to ER stress, amino acid biosynthetic genes are expressed.

*Navigating the Endoplasmic Reticulum: New Insights and Emerging Concepts DOI: http://dx.doi.org/10.5772/intechopen.105737*


#### **Table 1.**

*Diseases related to endoplasmic reticulum (ER) stress.*

#### *4.2.7 Role of ER stress in age-related diseases*

According to current studies, the process of aging and age-related disorders are interlinked with ER stress response [44]. With aging, the normal cellular functions are declined, particularly slow degradation of chaperones, which results in increased aggregation of misfolded proteins [45]. These misfolded proteins accumulated in various organs of the body, such as in case of Alzheimer's disease (AD), an inflammatory neurodegenerative disease. In AD brains, ER stress responses have been observed, because ER is the site for synthesis of secretory and membranous proteins [46].

Aging disrupts the balance between UPR and pro-apoptotic signaling, leading to reduced protective response against ER-stress signaling [47]. Essential chaperons and enzymes, required for protein folding are functionally damaged with aging [48]. ER structure is also altered with aging. Hinds and McNelly reported dispersion in the highly organized ER cisternae [49].

Autophagy, a process activated by UPR system, remove the aggregation of misfolded proteins. Nevertheless, this process slows down with age, leading to neurodegeneration [50]. ER stress responses have been associated with certain neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, ALS and Huntington's disease. In neurodegenerative disorders, UPR activity is sustained, while apoptotic pathways are upregulated, encompassed by accumulation of aggregated proteins, hence neuronal death in old age [51].

It has been postulated that ER stress also plays role in the onset of metabolic disorders. For instance, in Type 2 Diabetes, a metabolic disease, caused by insulin resistance is regulated by various ER stress response mechanisms. The two main mechanisms that disrupt insulin activity are interlinked with ER stress [52].

#### **5. Conclusion**

ER is a complex and well-organized organelle, crucial for various cellular metabolic functions. ER homeostasis is maintained by a network of signaling pathways, collectively known as ER stress response, in order to deal with genetic, infectious and inflammatory stressors. With age, UPR, ER stress response mechanism, lost its activity thus less efficiently respond to these stressors. This results in onset of various age-related metabolic

and neurodegenerative diseases. Advent of ER stress targeted therapeutics, particularly those improving protein folding and efficiency of associated regulatory mechanisms, promoting early detection of misfolded proteins could be useful in preventing and treating age-related disorders discussed in this chapter. Moreover, detection of anomalies in the ER stress response may led to development of therapeutics that could maintain ER homeostasis. This represents so far unexplored approach for disease prevention.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Sikander Ali1 \* and Maria Najeeb2

1 Institute of Industrial Biotechnology, GC University, Lahore, Pakistan

2 Institute of Microbiology, UVAS, Lahore, Pakistan

\*Address all correspondence to: dr.sikanderali@gcu.edu.pk

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Navigating the Endoplasmic Reticulum: New Insights and Emerging Concepts DOI: http://dx.doi.org/10.5772/intechopen.105737*

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#### *Updates on Endoplasmic Reticulum*

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#### **Chapter 2**

## Endoplasmic Reticulum: A Hub in Lipid Homeostasis

*Raúl Ventura and María Isabel Hernández-Alvarez*

#### **Abstract**

Endoplasmic Reticulum (ER) is the largest and one of the most complex cellular structures, indicating its widespread importance and variety of functions, including synthesis of membrane and secreted proteins, protein folding, calcium storage, and membrane lipid biogenesis. Moreover, the ER is implicated in cholesterol, plasmalogen, phospholipid, and sphingomyelin biosynthesis. Furthermore, the ER is in contact with most cellular organelles, such as mitochondria, peroxisomes, Golgi apparatus, lipid droplets, plasma membrane, etc. Peroxisomes are synthesized from a specific ER section, and they are related to very-long-chain fatty acid metabolism. Similarly, lipid droplets are vital structures in lipid homeostasis that are formed from the ER membrane. Additionally, there is a specific region between the ER-mitochondria interface called Mitochondria-Associated Membranes (MAMs). This small cytosolic gap plays a key role in several crucial mechanisms from autophagosome synthesis to phospholipid transfer. Due to the importance of the ER in a variety of biological processes, alterations in its functionality have relevant implications for multiple diseases. Nowadays, a plethora of pathologies like non-alcoholic steatohepatitis (NASH), cancer, and neurological alterations have been associated with ER malfunctions.

**Keywords:** endoplasmic reticulum, mitochondria, peroxisomes, mitochondria, lipid droplets, MAM, phospholipid, lipid metabolism

#### **1. Introduction**

The endoplasmic reticulum (ER) is a dynamic organelle largely responsible for essential cellular functions. Its wide and diverse functionality transforms the ER into a key organelle in cellular stress, signaling, vesicle transport, and lipid homeostasis. The ER is often in a state of constant change, shifting its structure to promote cell adaptation to environmental changes. For this reason, ER mass or area can fluctuate depending on cellular state and conditions.

The ER membrane is a lipid bilayer comprising two compartments: a cytosolic region in contact with the cytoplasm and a luminal region, which is the space between the two ER membranes.

In addition, two very well differentiated structures can be found within the ER: the rough endoplasmic reticulum (RER) and the smooth endoplasmic reticulum (SER). These structures have a unique architecture that is specialized for different

cellular mechanisms. Specifically, RER is formed of flattened sheets and contains a large quantity of ribosomes, whereas SER has a more irregular construct, consisting of a tubular structure, and is lacking in ribosomes [1–3]. This indicates that RER is more associated with protein synthesis than SER and newly synthesized proteins in RER ribosomes could enter the RER lumen to achieve their final conformation. Therefore, in cell types that hold a high rate of protein synthesis, such as hepatocytes, it is essential that the RER is further developed [1, 4].

In contrast, SER has additional functions related to calcium storage, detoxification, lipid metabolism, steroidal hormones, and bile acid production. As a result, SER is more developed in cells with a high detoxification power, such as hepatocytes, or in both smooth and skeletal muscle cells (where SER is called sarcoplasmic reticulum). Further, since SER participates in lipid metabolism, its presence is required in adipocytes as well [1, 2, 5].

Additionally, there is a specific region within SER and RER called nanodomains, where molecules and proteins are grouped, harmonically working together to perform a precise function. Thus, proteins are not evenly distributed along with the ER but associated in clusters [6].

As mentioned, RER is specialized to accommodate both membrane and secreted protein synthesis, as well as post-translational modifications. In particular, this ER region is in contact with the nuclear envelope and allows the mRNA to enter from the nucleus to the RER lumen. The RER's involvement in post-translational modifications includes glycosylation (N-glycosylation), sulfurization, and correct protein folding. First, RER-directed proteins must be recruited via a specific signal peptide which is recognized by the ribonucleoprotein complex SRP (Signal Recognition Particle). Once this target signal is recognized, the protein synthesis is temporarily stopped, and the ribosome, mRNA, and the newly synthesized protein are transported to the RER membrane. Here, the complex interacts with a membrane receptor and the protein is translocated to the RER lumen. Nonetheless, the mRNA continues to be translated and, at this point, protein synthesis recommences. Finally, the process ends when the entirety of the protein is in the RER lumen, and its signal peptide is cleaved by a peptidase. In the lumen, some chaperones are present in order to avoid an incorrect folding of the newly synthesized protein. There are also a plethora of enzymes that modify proteins in this region, preparing them to be secreted, for instance [4, 7–10].

The ER has a "quality-control activity" that detects misfolded proteins. If the proteins cannot be successfully repaired, they are discarded and degraded. Specifically, misfolded proteins are detected by glucosyltransferase, which binds glucose to them. The bound glucose is recognized by calnexin, which attempts to correctly fold the protein on several occasions. When the problem is not solved, the misfolded protein is degraded through the proteasome, yielding amino acids that the cell can recycle into new proteins. However, when misfolded proteins reach relevant levels, they are detected by some sensors within the ER membrane. These sensors regulate cellular responses to ER stress in a process called UPR or unfolded protein response, and can imply both survival and non-survival pathways [11–13].

Despite the variety of functions of the ER, this chapter will primarily focus on two aspects: the ER's implication in lipid metabolism, transport, synthesis, and homeostasis, and the mechanism by which the endoplasmic reticulum interacts with other organelles to achieve this.

As stated above, the ER has specific regions of contact, termed membrane contact sites (MCS), with a multitude of cellular organelles. However, it should be noted that these interactions do not imply membrane fusion but classically they have been shown to be related to calcium flux regulation (**Figure 1**).

*Endoplasmic Reticulum: A Hub in Lipid Homeostasis DOI: http://dx.doi.org/10.5772/intechopen.105450*

#### **Figure 1.**

*Graphical scheme of endoplasmic reticulum interactions with peroxisomes, mitochondria, lipid droplets, plasma membrane, and Golgi apparatus. With mitochondria, ER works coordinately to synthesize some of the major phospholipids, including phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Contacts between ER and peroxisomes permit the synthesis of plasmalogen and cholesterol, while sphingomyelin (SM) is obtained as a result of interactions between ER and Golgi apparatus. Both peroxisomes and lipid droplets derive from ER membranes, implicating proteins such as Seipin and FIT (in lipid droplet biogenesis), or Pex3 and Pex19 (in peroxisomes biogenesis). However, ER can also interact with more organelles such as lysosomes. ER can also contact with plasma membrane; these contacts permit, for instance, the phosphatidylinositol (PI), phosphatidic acid (PA), PS, and phosphatidylinositol 4-phosphate (PI4P) transport, thanks to PITPNM1 and ORP5/8 action.*

Nevertheless, at present, MCS is also seen to be associated with lipid exchange, lipid synthesis (phospholipids, sphingomyelin, cholesterol, or plasmalogen biosynthesis, for example), and vesicle traffic [3, 14–16].

#### **2. Endoplasmic reticulum and mitochondria**

Mitochondria are known to be the powerhouse of the cell due to their energy production and homeostasis-related functions. For instance, some of the main processes in ATP obtention, such as the respiratory chain reactions or the Krebs cycle, occur in the mitochondria. Hence, mitochondria are especially essential in organs, tissues, or cell structures that require profuse amounts of energy, including the heart, neurons, or sperm flagella.

Interestingly, mitochondria can also enhance programmed cell death, apoptosis. Particularly, it intervenes in the intrinsic or cellular pathway, which can be activated by different cellular stimuli, including DNA damage, or ER stress. The process begins with the liberation of pro-apoptotic substances such as Cytochrome C, which participates in the respiratory chain. Cytochrome C then goes on to activate a signal cascade leading to cellular death. Clearly, mitochondria have an important involvement in some vital cellular functions, meaning its dysfunction is implicated in a large number of diseases, such as cancer, metabolic, neuronal, cardiovascular, or genetic disorders [17, 18].

Furthermore, mitochondria have unique features such as the presence of their own circular DNA as well as an inner and outer membrane. Additionally, they are highly dynamic organelles, being in a constant fusion and fission cycle based on cellular state and environmental stimuli.

Mitochondrial DNA (mtDNA) is constituted of two strands, heavy and light. The heavy strand is enriched in guanines, and codes for 12 subunits of the respiratory chain, 14 tRNAs, and two rRNAs. However, the light strand only codes for 8 tRNAs and one subunit. Despite that mitochondrial DNA does not code for a great quantity of RNAs or proteins, it is essential for good cell functionality [19–21].

Another important role attributed to mitochondria is phospholipid biogenesis, which takes place in a highly specialized association between the ER and the mitochondria called Mitochondria-Associated Membranes (MAM). Despite the fact there is currently no specified definition of exactly what MAMs are, it has been established that this area regulates processes such as apoptosis, lipid synthesis, transport, calcium homeostasis, autophagy, and mitophagy. MAMs structure is also required for phospholipids transport between the ER and the outer mitochondrial membrane (OMM) [15, 22–24].

Calcium is an essential regulatory element in mitochondria that regulates metabolism, apoptosis, and autophagy. In MAMs, calcium ion transfer to organelles is promoted due to its abundance of calcium transport channels. Specifically, mitochondria uptakes calcium ions through outer membrane voltage-dependent anion channels (VDAC) [25].

#### **2.1 Phospholipid synthesis: ER and mitochondria cooperation**

As previously explained, the endoplasmic reticulum and mitochondria coordinate together to synthesize some of the most important glycerophospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), which represents approximately 50% of total phospholipids located in cellular membranes [26].

Glycerophospholipids are the major component in cellular membranes, both intracellular (ER, mitochondrial, and peroxisomal membranes) and extracellular facing (plasma membrane). These lipids present a polar head (phosphate group) and a hydrophobic tail, formed by fatty acids. This duality gives them an amphipathic character, endowing their characteristics to the membranes. Moreover, the hydrophobic tail can be bound with choline, ethanolamine, and serine forming, PC, PE, and PS, respectively [27, 28].

In mammals, all glycerophospholipids are synthesized from a common molecule, diacylglycerol (DAG), which derives from phosphatidic acid. Throughout the synthesis process, a large number of enzymes and many intermediate molecules are generated. The vast majority of these molecules are in the ER or the mitochondrial membrane. The first step in glycerophospholipid synthesis is phosphatidic acid generation. Two acyltransferases (glycerol-3-phosphate acyltransferase-1, GPAT1, and acylglycerophosphate acyltransferase) located in the ER and outer mitochondrial membrane must act on a glycerol-3-P molecule. The phosphatidic acid-phosphatase 1 (PAP-1), a cytosolic enzyme activated upon contact with ER membrane, acts on phosphatidic acid-generating DAG. Finally, PC, PS, and PE are synthesized from DAG [29, 30].

Mammalian cells can synthesize phosphatidylethanolamine from phosphatidylserine. In order for this to occur, PS needs to be decarboxylated by the action of an inner

#### *Endoplasmic Reticulum: A Hub in Lipid Homeostasis DOI: http://dx.doi.org/10.5772/intechopen.105450*

mitochondrial membrane enzyme, PISD (mitochondrial phosphatidylserine decarboxylase). For PE formation, an alternative synthesis pathway is utilized, the Kennedy pathway. In this pathway, ethanolamine kinase phosphorylates ethanolamine, that comes from the extracellular environment via plasma membrane, and there is several intermediate enzymatic steps, leading to the formation of PE in the ER [31].

The Kennedy pathway is also used to obtain phosphatidylcholine. When choline is in the cytoplasm, it is phosphorylated by the choline kinase. Once phosphorylated, the choline-phosphate cyticylyltransferase catalyzes CDP-choline (cytidine-5-diphosphocholine) formation. Afterward, 1,2-diacylglycerol choline phosphotransferase, transfers a DAG molecule to CDP-choline, finally generating phosphatidylcholine in the ER [32].

In parallel, PE can also be converted to PC, but PE must first be methylated three times by phosphatidylethanolamine N-methyltransferase (PEMT), which is located in ER membrane. In general, this is not a representative pathway, except for in hepatocytes, where there are significant quantities of phosphatidylcholine produced [32, 33].

There are two enzymes found in MAMs that can synthesize phosphatidylserine: PSS1 (Phosphatidylserine Synthase 1) and PSS2 (Phosphatidylserine Synthase 2). PSS1 catalyzes the exchange of choline from phosphatidylcholine for serine, whereas PSS2 performs the equivalent exchange with ethanolamine from phosphatidylethanolamine. In both cases, phosphatidylserine is obtained [34, 35].

Recently, it has been described that Mitofusin 2 (Mfn2) participates in the phosphatidylserine transport between ER and mitochondrial outer membrane. Mfn2 is a GTPase protein located in the outer mitochondrial membrane, that classically, was associated with the process of mitochondrial fusion, regulating the fusion of two OMM [36].

Beyond controlling the mitochondrial fusion process, *Hernández-Alvarez et al.* demonstrated that the ablation of Mfn2 in mouse livers causes inflammation, triglyceride accumulation, fibrosis, and liver cancer with age. In addition, a reduction of Mfn2 levels has been observed in hepatic biopsies from patients with non-alcoholic

#### **Figure 2.**

*Graphical scheme of phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) biosynthesis in mitochondria-associated membranes (MAMs) in a healthy (left) and Mitofusin 2-ablated (right) liver. In ER, PC thanks to the phosphatidylserine synthase 1 (PSS1), is transformed to PS, which is transported to mitochondria due to Mitofusin 2 (Mfn2) action. There, PS is converted to PE, a process catalyzed by phosphatidylserine decarboxylase (PISD), which is in the mitochondrial membrane. Once PE is synthesized, it is transferred to ER, where is converted to PC or PS, depending on the enzyme implicated, phosphatidylethanolamine N-methyltransferase (PEMT) or phosphatidylserine synthase 2 (PSS2), respectively.*

steatohepatitis (NASH), a disease related to lipid metabolism. The levels of this protein were also lower in mouse models of steatosis or NASH. Furthermore, its reexpression in a NASH mouse model ameliorated the disease, therefore, demonstrating that Mfn2 protects against liver disease [15].

A probable explanation for the protective role of Mfn2 is presented in the same study; the authors demonstrated that Mfn2 has the ability to bind and help in the transfer of phosphatidylserine across ER-mitochondria contacts, generating PS-enriched domains. This facilitates PS transfer to mitochondria and further mitochondrial phosphatidylethanolamine synthesis. This transfer occurs in MAMs (**Figure 2**). Hence, a reduction of Mfn2 hepatic levels leads to poor PS transfer and phospholipid synthesis, causing ER stress, NASH-like phenotype, and liver cancer [15].

Additionally, Mfn2 deficiency alters PSS1 and PSS2 protein levels, inhibiting PS synthesis. This was also observed in the Mfn2 liver knock-out mouse model. The lack (or reduction) of Mfn2 also generates MAMs remodeling (altering the phospholipid composition in ER-mitochondrial contact sites). This modification leads to triglyceride accumulation, insulin resistance, and impaired phospholipid synthesis [15]. Other proteins associated with PS transport are oxysterol binding related proteins 5 and 8 (ORP 5/8) and synaptic vesicle membrane protein (VAT1) [24, 37]. ORP8 downregulation was also related to liver cancer [38]. However, whether PS deficiency is the cause, or the consequence needs to be further investigated.

#### **3. Endoplasmic reticulum and peroxisomes**

Peroxisomes are a highly versatile single membrane organelle present in many eukaryotic cells, including yeast. They can modify their morphology, size (0.2–1.5 μm), number, and activity depending on their nutritional state, cell type, or cellular environment. In mammals, peroxisomes contain a diverse range of enzymes making them organelles essential for several biochemical pathways. Some of the many roles of the peroxisome include fatty acid β-oxidation, bile acid synthesis, amino acid catabolism, polyamine oxidation, metabolism of reactive oxygen, and nitrogen species. Though all these functions are relevant, fatty acid β-oxidation is the most relevant. This process is critical for very-long-chain fatty acids (VLCFA) shortening, which mitochondria are not able to metabolize [39–41].

There are large amounts of oxidative enzymes contained within peroxisomes which can be observed in electron microscopy as crystals inside the organelle. Some of these enzymes include oxidase and catalase which use molecular oxygen to oxidize fatty and amino acids. Due to the high grade of toxicity within the peroxisome, catalase uses hydrogen peroxide (H2O2) to eliminate toxic/harmful substances, such as ethanol or methanol, or to oxidize new substrates. For this reason, peroxisomes are more abundant in cells undergoing detoxification processes, such as hepatocytes or kidney cells [39–42].

It has been described that peroxisomes interact with different organelles (lipid droplets, ER, mitochondria, lysosomes, etc.) through their contact sites to maintain lipid homeostasis and metabolism. For instance, peroxisomes transform VLCFA into medium-chain fatty acids, lipids that will be converted into water and CO2 by mitochondrial action. Alterations in peroxisomes are associated with several pathologies and rare genetic diseases, usually affecting the brain, kidney, liver, and skeletal muscle. In the brain, peroxisomes play a crucial role in the synthesis of plasmalogens, a phospholipid especially enriched in myelin. Any alteration in peroxisomes will lead to severe demyelination in neurons, causing the neurological

component observed in peroxisomal diseases. One example is the Zellweger syndrome, produced by a deficiency in peroxisomal biogenesis [39, 40, 42–44].

#### **3.1 Peroxisome biogenesis: ER role**

The peroxisomal membrane has a similar composition to the endoplasmic reticulum membrane. This provides a good understanding of the origin of peroxisomes; preperoxisomal vesicles in the ER (a specialized and delimited area of the ER). However, peroxisomes can also derive from another peroxisome through a process of fission [45]. During peroxisomal budding, a number of peroxisomal membrane proteins (PMP) are first directed to the ER, specifically in the specialized ER subdomain, from which pre-peroxisomes are budded. Pex3 and Pex19 are proteins that are particularly relevant in this process; Pex3 is a PMP located in ER membrane, while Pex19 is found in the cytosol. Pre-peroxisomal budding occurs due to an interaction between these two proteins [45, 46]. As the ER participates in peroxisomal synthesis and peroxisomes play a relevant role in lipid metabolism (plasmalogen and cholesterol synthesis), it could be suggested that the ER has an indirect function in all these processes.

#### **3.2 Peroxisome-ER coordination in lipid homeostasis: cholesterol and plasmalogens biosynthesis**

ER and peroxisomes work in coordination to maintain lipid homeostasis. There is an intimate relationship between these two organelles, transferring components and essential molecules to each other. For example, the ER transfers some important lipids to peroxisomes, whilst the ER receives some plasmalogens precursors from the peroxisome. Plasmalogens are ether phospholipids that represent approximately 20% of total phospholipids in humans. Synthesis of these molecules begins in peroxisomes and ends in the ER. As well as plasmalogens, peroxisomes can also synthesize cholesterol and part of its precursors. These precursors will then be transferred to the ER to complete their synthesis, demonstrating the complementary relationship between the ER and peroxisomes in their ability to synthesize cholesterol [39, 47, 48].

A major site of plasmalogens is in the nervous and immune system and heart; their main function is to protect these systems from oxidative damage produced by reactive oxygen species (ROS) or reactive nitrogen species (RNS). Synthesis of these molecules begins with the peroxisome phase, which initially involves esterification of dihydroxyacetone phosphate (DHAP) with an Acyl-CoA, catalyzed by DHAP acyltransferase (DHAP-AT). The resulting molecule, 1-acyl-DHAP, is then transformed to 1-O-alkyl-DHAP as a result of the action of alkyl dihydroxyacetone phosphate synthase (ADHAP-S), incorporating fatty alcohol and generating a fatty acid. Once 1-O-alkyl-DHAP is synthesized, it is transported to ER, where it will be transformed several times to obtain choline or ethanolamine plasmalogens (**Figure 3**). Hence, plasmalogen synthesis is another clear example of the relationship between peroxisomes and ER [39, 49, 50].

Nevertheless, the synthesis of this type of phospholipid is not the only process in which peroxisomes and the ER work together; a similar mechanism occurs with cholesterol. Principally, peroxisomes partake in the synthesis of farnesyl-pyrophosphate (FPP), an intermediate of terpenoid, terpene, and sterol biosynthesis; subsequently, the FPP generated is again transferred to the ER. Here, FPP experiences sequential modifications and finally results in the formation of cholesterol. Lastly, another means by which cholesterol synthesis can be initiated via peroxisomes is through

#### **Figure 3.**

*Graphical scheme of plasmalogen (left) and cholesterol (right) biosynthesis in peroxisome and endoplasmic reticulum. In plasmalogen synthesis, first, dihydroxyacetone phosphate (DHAP) is converted to 1-acyl-DHAP, a reaction catalyzed by DHAP acyltransferase (DHAP-AT), which incorporates an acyl-CoA molecule. Then, 1-O-alkyl-DHAP is obtained from 1-acyl-DHAP, through the incorporation of a fatty alcohol thanks to alkyl dihydroxyacetone phosphate synthase (ADAP-S). 1-O-alkyl-DHAP is transported to ER; there it suffers some modifications (not shown) until obtaining choline or ethanolamine plasmalogens. In cholesterol biosynthesis, the peroxisomal phase generates farnesyl-pyrophosphate (FPP), which is then transported to ER. There, FPP is modified (not shown), obtaining cholesterol. Mevalonate needed for FPP synthesis can be transferred from ER or be generated from acyl-CoA obtained during β-oxidation of very-long-chain fatty acids (VLCFA).*

Acetyl-CoA, derived from peroxisomal β-oxidation of very-long-chain fatty acids. Alternatively, it can continue synthesis from an intermediate, mevalonate, transferred from ER (**Figure 3**). In both cases, once FPP is obtained, cholesterol synthesis continues in the ER [47, 51, 52].

### **4. Endoplasmic reticulum and Golgi apparatus**

Golgi apparatus is an organelle with two main functions: post-translational protein modification and sorting, packing, routing, and recycling membrane proteins. In the Golgi complex, four regions can be distinguished: ER-Golgi intermediate complex, cis *Endoplasmic Reticulum: A Hub in Lipid Homeostasis DOI: http://dx.doi.org/10.5772/intechopen.105450*

and trans-Golgi network (the nearest and farthest cisternae to ER, respectively), and Golgi stack, which is divided into medial and trans compartments (corresponding to the central region of Golgi apparatus). Additionally, the Golgi complex has unique, biochemically distinct enzymes, that are distributed throughout its space [53, 54].

The newly synthesized proteins enter into the ER, where they are introduced into vesicles (they move from the ER-Golgi intermediate compartment to the cis-Golgi network). Finally, the vesicles reach the trans-Golgi network, which delivers these molecules to their target destinations. Despite the Golgi complex playing a fundamental role in protein transport and post-translational modifications, it is also involved in the synthesis of certain lipids, such as sphingolipids, and especially sphingomyelin, which is vital for correct cell functionality [53–55].

#### **4.1 Golgi apparatus-ER phospholipid transport: sphingomyelin synthesis**

Sphingomyelin is mainly located in the outer monolayer of the plasma membrane and is crucial for the functioning of a number of cellular processes, such as immune recognition, cell differentiation, growth, and apoptosis. Furthermore, sphingomyelin is known to be a major component of the myelin covering certain neuron axons. Namely, it binds hydrocarbon chains, improving myelin strength [56].

It is synthesized largely from ceramide and phosphatidylcholine, which are lipids obtained in the ER. Sphingomyelin synthase-1 (SGMS1), the enzyme required to synthesize sphingomyelin, is localized in the Golgi apparatus, thus its precursors must be transported from the ER to the Golgi complex.

Sphingomyelin can be obtained both in the plasma membrane and in the Golgi apparatus. Nonetheless, sphingomyelin synthesis is residual in the plasma membrane and this reaction is catalyzed by a different enzyme, sphingomyelin synthase-2 (SGMS2) [30, 57, 58].

As mentioned before, ceramide and phosphatidylcholine must be transferred from ER to the Trans Golgi network (TGN) to synthesize sphingomyelin, a process that occurs in the MCS. Ceramides are primarily located in ER membrane due to their low hydrophilicity. Moreover, this lipid can be transported to Golgi apparatus through one of two mechanisms: coatomer-dependent vesicular transport or action of a cytosolic peptide, the ceramide transfer protein (CERT). CERT transport is regulated by phosphatidylinositol-4-phosphate (PtdIns(4)P) quantity in the TGN. Once sphingomyelin is synthesized, it is transported to the plasma membrane via vesicular transport [30, 57, 58].

#### **5. Endoplasmic reticulum and lipid droplets**

Lipid droplets (LD), also known as adiposomes, are a spherical cytosolic organelle that stores triglycerides and cholesterol esters, providing an energy reserve. They are found in animals, plants, fungi, and in some bacteria. They comprise two different structures: a hydrophobic core formed by neutral lipids, and a polar phospholipid monolayer containing proteins (such as perilipin, PLIN), which partially regulate their functions. This ER-derived organelle plays a considerable role in lipid and energy homeostasis. For example, lipid droplets can generate contacts with a multitude of organelles, including mitochondria, peroxisomes, lysosomes, and the ER, allowing the transfer of lipids between them [59–61].

LD also seem to have a relevant role in infections where in some viral, fungal, or bacterial infections, microorganisms use LD in their cycle of infection. Examples that illustrate this can be seen in the case of some viruses, which exploit lipid droplets to assemble inside the cell or, on the other hand, mycobacteria and other intracellular pathogens, which steal the lipids contained in these structures to adapt themselves to the cellular environment [62]. As well as this, LD can also regulate cellular toxicity, accumulate toxic lipids, and protect vital structures from oxidative stress. Therefore, alterations in their function or physiology trigger diseases, such as NASH, obesity, or diabetes [59, 63, 64].

Depending on the cellular type, lipid droplets present distinct functionality. For instance, in adipocytes these droplets store triglycerides, waiting to be hydrolyzed when peripheral tissues require more energy. In testis, ovaries, or suprarenal glands, the lipid droplets are smaller than adipose tissue and they accumulate cholesterol esters, necessary for steroid and sex hormone synthesis. Finally, in the liver, adiposomes facilitate lipoprotein synthesis (low-density lipoprotein, LDL, and highdensity lipoprotein, HDL). In mammals, tissues such as the liver, adipose tissue, and muscle contain an abundance of lipid droplets.

In addition, lipid droplet quantity is tightly associated with the nutritional status, metabolism, and nutrient availability of individuals. When there is an excess of nutrients, they are stored in lipids, increasing the number and size of lipid droplets. However, during starvation or nutrient depletion, lipids are mobilized, to synthesize the essential phospholipids or produce the required energy. Thus, lipid droplets are smaller and less abundant during this situation, especially in white adipose tissue and the liver [59, 60, 64, 65].

#### **5.1 Lipid droplets biogenesis: ER role**

As already explained, lipid droplets derive from a specific area of the ER membrane. Not only does the ER regulate and allow their genesis, but also has an important implication on lipid storage and metabolism (**Figure 4**).

Lipid droplets mainly store triglycerides and sterol esters, which are synthesized by enzymes mostly located in ER membrane. Prior to its incorporation into the lipid droplet, fatty acids are esterified with a sterol (to obtain a sterol ester) or diacylglycerol (to obtain a triglyceride). These reactions are catalyzed by acyl-CoA: cholesterol O-acyltransferases (ACAT1 and ACAT2), and diacylglycerol acyltransferases (DGAT1 and DGAT2), respectively. When the quantity of these lipids is significantly high, they tend to be grouped, forming what is called a "lipid lens" in the ER bilayer. It is thought that there are no proteins related to the formation of this lipid droplet precursor, due to a purely physical effect driven by their hydrophobicity characteristic. As more neutral lipids are synthesized, the lipid lenses tend to expand, eventually causing the lipid droplet to bud from ER membrane [66–69].

Unlike in secretory vesicles, lipid droplet biogenesis does not appear to require coat proteins, whereas the phospholipid composition of the ER membrane would be crucial. In particular, phospholipids can influence the membrane surface tension, which is extremely important to the rounded shape purchase of LD. During this process, the neutral lipid area in contact with the aqueous media is reduced maximally and is determined by the phospholipid type dominance. For instance, while phospholipids, such as PE hinder biogenesis, lysophospholipids enhance it. These aspects also determine the gemmating direction, releasing lipid droplets into the cytosol, although they can be eventually released into the ER lumen [70–72].

Nonetheless, it has been shown that some ER membrane located proteins (storage-inducing transmembrane, FIT) regulate lipid droplet budding. More

#### **Figure 4.**

*Graphical scheme of lipid droplet biogenesis from endoplasmic reticulum membrane. First, cholesterol esters and triglycerides (TAG) accumulate near the enzymes responsible for their synthesis; acyl-coenzymeA: Cholesterol acyltransferase 1/2 (ACAT1/2), and diacylglycerol acyltransferase 1/2 (DGAT1/2), respectively. This accumulation forms a lipid lens, a lipid droplet precursor. Lipid lens will grow, deforming ER membrane, a process promoted by certain phospholipids, such as lysophospholipids. To stabilize lipid droplet-ER contacts, Seipin is needed, helping in lipid droplet budding. Finally, when the lipid droplet formed has a correct size, it is released into the cytosol.*

specifically, FIT proteins maintain the ER lipidic composition and shape. In the absence of FIT, the quantity of sterol esters and triglycerides in the ER membrane increases through inhibition of lipid droplet gemmation. FIT seems to act by interacting directly with DAG; hence, a lack of FIT provokes DAG accumulation in ER, which inhibits the LD budding by altering ER morphology and membrane surface tension [73, 74].

In addition, the protein Seipin is also involved in the regulation of the formation of lipid droplets. This protein has important implications in stabilizing the contact sites between the ER and LD. Furthermore, when Seipin is absent, LD formation is delayed, meaning less incorporation of lipids and proteins, and so this leads to LDs generated becoming morphologically aberrant. Moreover, a protein related to peroxisomal biogenesis, Pex30, has been described to interact with Seipin in yeast. While in normal conditions Pex30 is located along the ER membrane, in Seipin mutants, it accumulates in LD biogenesis sites. When both Seipin and Pex30 are depleted, there is no LD biogenesis, neutral lipids are accumulated in ER membrane, peroxisomes are not well synthesized, and membranes present a phospholipid disbalance, increasing PC, phosphatidylinositol, and DAG. It is observed that Pex30 works in coordination with Seipin, controlling ER membrane lipid composition, especially in LD budding areas [75–77].

Once gemmated, lipid droplets will grow through different mechanisms that include lipid transfer from ER, LD fusion, or lipid synthesis in their membrane. Triglycerides can be synthesized in LD membranes due to the transfer of specific enzymes from ER. Moreover, ER supplies all the required phospholipids for the growth to LD [78, 79].

#### **6. Endoplasmic reticulum and plasma membrane**

The plasma membrane (PM) is a phospholipid bilayer that isolates the cell content from the outside. Although PM is mainly constituted of phospholipids (such as PC, PE, and PS), it also presents cholesterol, sphingolipids, and a huge variety of proteins, which allow the signal transduction. Furthermore, PM can establish contacts with ER where phospholipid and sterol transport occur [80, 81].

#### **6.1 Plasma membrane-ER phospholipid transport**

Contacts between ER and plasma membrane are mostly related to phospholipid synthesis and its signaling. For instance, during growth factors stimulation, cells translocate PITPNM1 (membrane-associated phosphatidylinositol transfer protein 1) from the Golgi apparatus to the plasma membrane. Specifically, this lipid transfer protein transports simultaneously phosphatidic acid from PM to ER and phosphatidylinositol from ER to PM. From phosphatidic acid and phosphatidylinositol many phospholipids with regulatory functions, such as phosphatidylinositol 4-phosphate (PI4P), DAG, or phosphatidylinositol 4,5-biphosphate (PI4,5P2), can be synthesized and serve as precursors for the synthesis of PC, PS or PE. During high glucose concentration in pancreatic β-cells, it is observed another protein (phospholipid transfer protein C2CD2L, also known as TMEM24) with a similar role to PITPNM1 (it transports PI from ER to plasma membrane) [80, 82].

On the other hand, more proteins regulate lipid transport between ER and plasma membrane. For instance, ORP5/8 can transfer PS from ER to plasma membrane and PI4P in the opposite direction [80, 82]. All this continuous transport between these two organelles allows cells to main the main phospholipids levels and allows their signaling, which is essential in external and internal factors stimulation.

#### **7. Main age-related alterations in ER and MCS**

It was observed that in senescence cells, the endoplasmic reticulum increases its size, also altering its functionality. Moreover, there is variation in calcium concentration and their proteins, including chaperones, and glycosyltransferases. This alteration leads to a reduction of protein folding efficiency and an increase in misfolded protein accumulation, generating a long-term unfolded protein response. This permanent UPR activation forces cells to cell death and abnormal mitochondrial calcium signaling [83–85].

With age, alterations in MAMs, observed as an increase in the distance between ER and mitochondria, have also been seen, indicating its role in pathological conditions associated with age. This condition impairs calcium signaling and autophagy and increases ER stress. In fact, MAMs alterations have been reported in several age-related diseases, including cardiac cell senescence, cancer, inflammation, and metabolic diseases [83–85].

Apart from ER-mitochondria interactions, other types of relationships such as ER-plasma membrane, ER-Golgi, ER-lipid droplets, ER-lysosomes, ER-peroxisomes are interesting to explore in order to find out what occurs with aging.

*Endoplasmic Reticulum: A Hub in Lipid Homeostasis DOI: http://dx.doi.org/10.5772/intechopen.105450*

#### **8. Conclusions**

The endoplasmic reticulum plays a central role in lipid homeostasis due to it establishing contact with essentially all cellular organelles, including mitochondria, peroxisomes, Golgi apparatus, lipid droplets, and plasma membrane (**Figure 5**).

To synthesize some of the more abundant glycerophospholipids, coordinated action between ER and mitochondria is needed, implicating several enzymes located in both organelles.

ER is also related to the biogenesis of peroxisomes and lipid droplets; peroxisomes interaction with ER can also generate cholesterol, essential in the plasma membrane fluidity maintenance, and plasmalogens, that protect cells from oxidative damage. As far as lipid droplets are concerned, they are generated from specific regions of the ER and their main function is to store triglycerides and cholesterol esters.

ER can also interact with the plasma membrane, exchanging phospholipids such as acid phosphatidic and phosphatidylinositol, leading to correct cellular signaling and response to extracellular stimuli. Finally, the endoplasmic reticulum also contacts the Golgi apparatus, an important event in sphingomyelin biosynthesis.

With age, it is documented that alteration in ER size and functionality, leading to a chronic UPR activation, induces apoptosis and aberrant calcium signaling. Moreover,

#### **Figure 5.**

*Scheme of endoplasmic reticulum contacts with multiple cellular organelles, such as mitochondria, peroxisomes, Golgi apparatus, lipid droplets, and plasma membrane; and, its functions.*

in aging models it was an increase in ER-mitochondrial distance was also observed, also altering the ER-mitochondria communication. However, the consequences of age in the other ER contacts nowadays are unclear.

Although we have explained some of the ER interactions, there is not enough information on all the synergistic functions that the ER has. Further research is needed.

#### **Acknowledgements**

M. I. H-A acknowledges the support from the "Ayudas para contratos Ramón y Cajal (RYC) 2018" (RYC2018-024345-I) from the "Ministerio de Ciencia e Innovación" from Spain. We thank Inma Martínez-Ruiz and Alysha Jiwa for comments and the critical reading of this manuscript.

### **Funding**

This study was supported by research grants from MICINN (PID2019- 105466RA-I00 AEI/ 10.13039/501100011033 and RYC2018-024345-I MCIN/AEI/ 10.13039/501100011033) and "la Caixa" Foundation project HR21-00430.

### **Author details**

Raúl Ventura1 and María Isabel Hernández-Alvarez1,2,3\*

1 Facultat de Biologia, Departament de Bioquímica i Biomedicina Molecular, Universitat de Barcelona, Barcelona, Spain

2 Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain

3 Institut de Biomedicina de la Universitat de Barcelona IBUB, Barcelona, Spain

\*Address all correspondence to: mihernandez@ub.edu

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Endoplasmic Reticulum: A Hub in Lipid Homeostasis DOI: http://dx.doi.org/10.5772/intechopen.105450*

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#### **Chapter 3**

## Responses of Endoplasmic Reticulum to Plant Stress

*Vishwa Jyoti Baruah, Bhaswati Sarmah, Manny Saluja and Elizabeth H. Mahood*

#### **Abstract**

Global climate change has resulted in alterations in the biotic and abiotic conditions of the planet. This has led to changes in the agricultural system resulting from reduced water availability, increased temperature increase in the population and occurrences of pests and diseases. Plants are adversely affected when they experience any stress retarding their growth, development and productivity. Endoplasmic Reticulum (ER) is an organelle that shows a tremendous response when subjected to stress conditions. Therefore, to explore and comprehend plants' multidimensional interactions when subjected to stress conditions, an insight into the molecular stress signalling in the ER in response to the stress situation is discussed in this chapter.

**Keywords:** biotic stress, cold stress, drought, endoplasmic reticulum, heat stress, salt stress, plant defence

#### **1. Introduction**

The endoplasmic reticulum (ER) is a versatile, dynamic and largely pleiotropic subcellular organelle forming an essential part of eukaryotes. It is one of the largest in size, complex in functionality and variable in architecture [1]. It plays a significant role in maintaining the spatial organisation of endomembrane organelles by acting as an architectural framework. It also synthesises essential cellular building blocks like lipids and proteins. ER lies at the core of the endomembrane system, which comprises unified endocytic and biosynthetic cellular processes and is composed of two different structural subdomains. One is the nuclear envelope enclosing the nucleus, and the other is the peripheral ER comprising the interconnected network of flattened sacs and tubules [2, 3]. At a submicron level, the ER network is organised in morphologically distinct domains that assume specific functions [4]. The ER represents the organelle with the largest membrane surface area owing to its network of interconnected tubules and flattened cisternae. Several genetic studies aided with live-cell imaging have illustrated the underlying drivers and the implausible dynamism of ER. The ER exemplifies a secretory pathway gatekeeper that controls protein quality control, its folding, signalling, and degradation across multiple checkpoints and impacts the general plant growth cellular homeostasis.

The ER is responsible for synthesising an array of proteins such as enzymes, ion channels, receptors and cargo molecules that modulate several essential physiological processes, which are ultimately either retained in or distributed from the ER [5–7]. Besides syntheses, ER also serves as a storehouse for carbohydrates and calcium [8, 9] and plays an essential role in abiotic and biotic stress resistance through the control of protein folding and signalling [10]. These characteristics of ER make it a functionally and structurally non-uniform yet morphologically continuous cellular compartment.

#### **2. ER in plants: structure and function**

In plant cells, the ER plays a critical role in the organism and bears a strong connection with other plant organelles like the vacuole, Golgi apparatus [7, 11, 12] and chloroplast [13, 14] forming the shape of a spider-webbed membrane network and any defect in its functionality can give rise to several developmental defects [15–18]. Based on electron microscopy studies, the ER can be differentiated into smooth ER-bearing subdomains with associated ribosomes, rough ER with subdomains of ribosomes-free regions, and nuclear envelope regions which are enwrapped by the ER forming a double membrane demarcating the nucleus [4, 19, 20]. The ER forms connections with the neighbouring cells through channels called plasmodesmata that protrude into the cell wall and interconnect with the cytoplasm of the neighbouring cells [21, 22], forming a unique organelle that cannot be delimited by a cell boundary. Electron microscopy-aided studies have also reported that the plant ER form connections with other membranes, including vacuoles, plastids, mitochondria and Golgi apparatus. In addition to the ER- plasma membrane contact sites (EPCSs) and plasmodesmata [4, 23]. The optical trapping and tweezer system has established physical contact of the chloroplast and Golgi apparatus with the ER [24, 25].

It has been thought that the movement of the ER influences the movement of other organelles, which is possible by the several network connections from the ER to the other cell organelles. While the ER enlarges, contracts or reorganises its morphology, its integrity is maintained by unfolded protein response (UPR) [26] signalling demonstrating the relationship between the ER morphology and its functional integrity [27–29]. Therefore, the connection of ER with other cellular organelles and compartments is crucial for the exchange of constituents as well as for their function and spatial distribution [29, 30].

The most fascinating feature of the ER in the plant cell is its extraordinary dynamicity which can be easily observed through microscopic analysis resulting in the overall evolving architecture of the tubules and cisternae of the ER that are in a state of continuous movement and rearrangement [27, 29, 31]. This morphological reorganisation of the ER has been reported previously where in the initial phases of cell growth, the ER was observed as a compact entanglement of membranes which undergo reorganisation into an open network as the cell growth advances [27, 32]. The ER organisations become even more complicated in root cells where the ER assumes a condensed form, primarily where the root hairs originate [27, 33]. Evidence of a link between ER shape and ER function can be demonstrated when mutations in Root Hair Defective3 (RHD3) ER-shaping protein compromise the functional organisation of the ER by elongation of the cells and transitioning to a reticulate pattern from a more sheet-like form [29, 30].

#### **3. Response of plant ER to stress**

The changing climate poses a significant risk to the plants owing to the transitions in its natural environment from typical to very harsh conditions. When plants are exposed to adversities like low water availability, high or chilling temperature, salt concentrations in the soil or pest and disease infestations, they usually respond by changing their response in a dynamic way. Although several studies have aimed to investigate the response of plants to individual environmental stress, in the field, the scenario is such that the plants may be exposed to multiple stresses, and their response may be quite different from that in the laboratory [34]. These dynamic responses in plants include changing the proteome by changing the gene expression patterns [35]. In plants, the ER is the prime organelle that regulates the stress responses [36, 37], as any stress which affects protein folding leads to a cellular homeostatic response mediated by the UPR in response to ER stress [36, 38]. ER is the point of synthesis of the majority of plant cell proteins and also the point of folding of unfolded or misfolded proteins, which are aided by chaperons and co-chaperons [39, 40]. The ER quality control (ERQC) and ER-associated degradations (ERAD) are two mechanisms that maintain the folding of proteins and degrade the misfolded proteins, respectively [39]. Nevertheless, sometimes, these mechanisms are surpassed by the UPR when there is an accumulation and persistence of misfolded and/or unfolded proteins leading to a state where the organelles trigger specific signalling pathways to restore the ER mechanism and recover from the stress [41].

#### **4. ER stress signalling in response to heat stress**

Heat stress often poses a serious threat to plants, as it negatively affects photosynthesis and fertility and can at times result in death. At the cellular level, symptoms of heat stress include the accumulation of misfolded or denatured proteins, production of ROS, microtubule disorganisation, and membrane instability. As the endoplasmic reticulum (ER) is the site of production for many proteins and lipids, this organelle is tightly linked with the heat stress response. While heat stress response mechanisms include the accumulation of osmolytes and antioxidants, perhaps the most canonical response is the production of Heat Shock Proteins (HSPs): molecular chaperones which facilitate proper protein folding and/or disaggregation of misfolded proteins [42].

Two critical components of the ER's Unfolded Protein Response (UPR) are also the major regulators of the heat stress response: the transcription factor bZIP28 and cytosolic RNA-splicing enzyme IRE1. bZIP28 was first determined to be involved in the heat stress response in *Arabidopsis* through a combination of co-expression analysis, cellular localisation studies, and reverse genetics [43]. Further studies have elucidated the mechanism of action of bZIP28 in the heat stress response [44, 45]. Briefly, in unstressed cells, bZIP28 is tethered to the ER membrane by an HSP (Binding Immunoglobulin Protein, or BiP), but when misfolded or denatured proteins accumulate in the ER, BIP is competed away from bZIP28, thereby releasing it to act as a transcription factor and upregulate the expression of other HSPs. *Arabidopsis* IRE1 was found to upregulate bZIP60 – another transcription factor upregulating stress-responsive genes – through cytosolic mRNA splicing [46]. These proteins are at once essential for the UPR stress response in general and the heat stress response, specifically as HSPs are among their direct or indirect targets and their loss of function

results in decreased survival under heat stress [47]. These initial studies uncovered the ER's direct role in mediating heat stress.

More recent studies have discovered roles for the ER in the metabolism under heat stress, specifically in the starch and lipid biosynthesis pathways. Although the role of the ER in starch metabolism is less clear than its role in lipid metabolism, several studies have implicated its involvement. In rice, heat stress during grain set can result in a "chalky" grain appearance and decreased starch content. Several studies have found altered expression of starch biosynthetic genes in mutants of the UPR, particularly in seeds [48, 49]. Other studies have found that the application of heat stress during the pre-cellularisation stage of grain set results in increased chalkiness and decreased starch levels [50]. A final study found that components of the UPR regulate starch content and chalkiness in rice seeds under heat stress [51]. When considered together, these results implicate that the ER/UPR regulates starch metabolism under heat stress. While the exact mechanisms underpinning this regulation are unclear, it has been known for some time that the ER is a site of lipid biosynthesis in plants. Tight control of lipid metabolism under heat stress is required as plants alter the lipid composition of membranes in order to combat heat-induced membrane instability.

In many plants, including *Arabidopsis* [52], tomato [53], wheat [54], and turfgrasses [55], an increase in the amounts of ER-produced lipid classes occurs under heat. Forward genetic studies have further demonstrated that ER stress, as well as heat stress, can alter lipid metabolism. For example, *Arabidopsis* mutants with inactive Fatty Acid Desaturase 2 (fad2 mutants) show either aberrant phenotypes or increased symptoms under heat [56] or ER stress (imposed by tunicamycin) [57], respectively, compared to WT. In soybean, isoforms of FAD2 were found to be unstable in high temperatures [58], and FAD2 transcripts were found to be downregulated under heat stress in *Arabidopsis* [59]. These results suggest that lipid metabolism, and to some degree starch metabolism, is dependent upon favourable conditions in the ER and is altered under heat stress.

One of the open questions in ER stress regulation is how the stress is perceived by the ER and what molecules or phenomena serve as the stress signal(s). Extracellular Ca2+ was one of the first candidates hypothesised to be the cellular signal for heat stress [60]. Seminal studies hypothesised the following signalling response: the influx of extracellular Ca2+ via Cyclic Nucleotide Gated Channels activates Calmodulin, which activates kinases, which finally activates Heat Shock Transcription Factors [61]. Other early putative signals included a nuclear-localised histone sensor and two ER-localised unfolded protein sensors [61]. More recent studies have implicated other molecules to act as a signal for heat stress, specifically Jasmonic Acid (JA) and phytochromes. In a recent study in rice grains, JA biosynthesis and signalling activity was enriched among genes upregulated one hour after heat shock, and JA accumulated three hours after heat. Furthermore, treatment with Methyl JA (MeJA) increased the abundance of IRE1-spliced OsBZIP50(s), an ER-stress response biomarker [50]. In another study conducted in *Arabidopsis* seeds, HY5, a component of light signalling that is downstream of phytochromes, was found to negatively regulate the UPR [62]. Taken concurrently with evidence that phytochromes have thermo-sensing capabilities and that null phytochrome mutants exhibit a constitutive heat stress phenotype [63], the above study provides evidence that phytochromes may sense and transmit the heat stress signal to the ER. Further research is needed to determine the conditions under which these signals are active and if they act in conjunction with each other.

Heat stress can be particularly damaging to anthers and to the developing pollen. Recent evidence points to the UPR being constitutively active in male reproductive

*Responses of Endoplasmic Reticulum to Plant Stress DOI: http://dx.doi.org/10.5772/intechopen.106590*

tissues [64], and therefore it is critical to understand how the UPR is affected by heat stress in these tissues. In a recent study, several ER-stress genes, including BiP, other chaperones/HSPs, and genes involved in the ER protein cleaning system, were all upregulated under heat stress in pollen germinating *in vitro* [65]. Furthermore, through forward-genetic studies, it was recently discovered that ER-localised chaperones are critical for proper pollen development and seed set under heat stress. *Arabidopsis* mutants of the Thermosensitive Male Sterile 1 (TMS1) gene, encoding an HSP40, showed reduced fertility and pollen coat abnormalities when grown under 29°C [66]. Similarly, *Arabidopsis* mutants lacking IRE1 were male sterile at 29°C yet had viable pollen when grown at room temperature [64]. As proper protein production and secretion are critical for pollen tube growth, any disruption to this process – such as that caused by heat stress – is hypothesised to be detrimental to pollen growth and viability and, therefore, negatively affect fertility.

#### **5. ER stress signalling in response to drought stress**

Drought stress is a major driver of yield losses – with a tremendous potential to limit plant growth than any other abiotic stress [67]. An important drought response mechanism is the closure of stomata. This at once conserves water by decreasing evapotranspiration and lowers photosynthesis rates, leading to decreased growth rates. If a period of drought occurs during the reproductive or grain filling stage, yield losses can occur due to pollen sterility or embryo abortion. Plant responses and adaptions to drought stress include the aforementioned stomatal closure, deeper root growth, reduced leaf size and altered leaf orientation. At the cellular level, these drought responses result in loss of turgor, which stimulates the production of the hormone Abscisic acid (ABA). ABA is a canonical "stress response" hormone [68], bringing about morphological changes through the following signal transduction cascade: ABA molecules bind to their receptors, which causes inhibition of PP2C phosphatases, leading to the autophosphorylation and activation of SUCROSE NONFERMENTING1-RELATED SUBFAMILY 2 (SnRK2) kinases, which phosphorylate ABA-responsive transcription factors (AREBs), ultimately leading to the expression of stress-responsive genes.

Some abiotic stresses, notably salt and heat stress, stimulate ER stress by markedly increasing the numbers of misfolded proteins in a cell. However, the mechanism by which drought stress is related to ER stress has yet to be determined. Toward this end, several studies have found multiple components of the ER protein folding machinery to be essential for proper drought response, including several E3 ubiquitin ligases, the transcription factor bZIP60, and Binding Protein (BiP). BiP is a molecular chaperone that contributes to proper protein folding, especially under ER stress. Overexpression of BiP has been found to confer drought tolerance - specifically less stomatal closure, less wilting, and maintenance of turgor - in *Nicotiana tabacum* (tobacco) and *Glycine max* (soybean) [69, 70]. Interestingly, the drought tolerance was concurrent with a decrease in the levels of typical drought stress response mechanisms, including the osmolytes proline/sucrose/glucose, root biomass, and drought stress response genes NAC2, glutathione-S-transferase, and antiquitin [69]. Similar to BiP, E3 ligases play essential roles in ER protein folding. The putative E3 ubiquitin ligase SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) is homologous to the mammalian TEB4, a component of the ER-associated degradation pathway (ERAD) that marks non-functional proteins for destruction [71]. Both TEB4 and *Arabidopsis* SUD1 are implicated in the

regulation of sterol biosynthesis [71, 72] – an interesting link between the ER and cellular stress response as sterols are components of lipid membranes that can change membrane fluidity as the temperature fluctuates. Notably, mutations in SUD1 were found to restore drought hypersensitive2 (dry2) [72] – which show increased sensitivity to drought stress due to abnormal sterol composition in roots and aberrant ROS signalling [73] – to WT phenotypes, revealing a link between the ERAD and drought responses. Two additional E3 ubiquitin ligases, Rma1H1 (from *Capsicum annum*) and Rma1 (from *Arabidopsis*), confer drought tolerance when overexpressed, putatively through suppressing the trafficking of the aquaporin channel PIP2;1 [74]. The transcription factor bZIP60 – the target of the mRNA splicing enzyme IRE1 – is a third component of the ER stress response that confers drought tolerance when overexpressed. bZIP60 from *Boea hygrometrica* (the extremely drought-tolerant "resurrection plant") conferred drought tolerance to *Arabidopsis* when overexpressed through the up-regulation of several ER quality control genes [75]. These results, when considered together, suggest that increased activity of the ERAD provides enhanced drought tolerance and therefore that ER functions are essential for survival under drought stress, though further research is needed to pinpoint the specific functions of the ER components under drought stress.

Several studies have uncovered an interesting link between ER stress and ABA signalling under drought stress – suggesting that ER functions are mechanistically related to drought stress through ABA. One study mentioned above transformed bZIP60 from *B. hygrometrica* (BhbZIP60) into *Arabidopsis*, overexpressed it, and found enhanced drought tolerance and increased expression of both ER components and ABA-responsive genes [75]. Interestingly, the authors found that BhbZIP60 was able to bind to the ERSE cis-regulatory elements present in ER stress-responsive genes but unable to bind to ABA-responsive cis-regulatory elements. Further, BhbZIP60 (in its native host *B. hygrometrica*), was found to be highly upregulated under drought but not so under heat or salt – which highly upregulates bZIP60 expression in *Arabidopsis* [46]. Another study found that inactivating the ERSE in *Zea mays* PP2C-A gene – which encodes a phosphatase with important roles in ABA signal transduction – confers drought stress tolerance [76]. While the WT (ERSE inactive) promotor was responsive to only ABA, the mutant promotor (with a full, active ERSE) was responsive to ABA and ER stress signalling. From these results, the authors proposed the following mechanism for interrelating ER stress with drought stress. When drought stress occurs, it activates ABA signalling, which includes a feedback mechanism for tightly regulating levels of PP2C-A and thereby confers drought tolerance. However, drought stress also activates ER stress signalling, and when PP2C-A is also upregulated by ER stress, the feedback loop is broken, causing hypersensitivity to drought. Further research is needed to see if this is indeed the mechanism by which ER components are interrelated to drought stress, though multiple lines of evidence have implicated ABA signalling in this mechanism.

#### **6. ER stress signalling in response to salt stress**

In addition to serving as a site for protein synthesis and transport, ER also plays a crucial role in protein homeostasis under environmental stresses [77]. Stress-induced cellular disruptions such as improper protein folding, excessive protein degradation,

#### *Responses of Endoplasmic Reticulum to Plant Stress DOI: http://dx.doi.org/10.5772/intechopen.106590*

accumulation of unfolded protein or overexpression of a protein or increase in the signalling molecules induce ER stress [47].

Three interactive mechanisms, namely, ER quality control (ERQC), ER-associated degradation (ERAD) and the unfolded protein response (UPR) or ER-nucleus signalling pathway, have been described in relation to ER proteostasis function. Salt stress results in the accumulation of misfolded proteins leading to increased transcription of ER-localised proteins, including chaperons such as binding protein (BiP) [78] and Calreticulin (CRT) which aid in restoring proteostasis [79]. On accumulation of misfolded proteins, BiP physically binds to the misfolded proteins, prevents their aggregation and activates UPR signalling [80]. Overexpression of BiP has been reported to enhance salt stress tolerance. Wang et al. [78] identified *BiP* in Capsicum annuum L. and observed increased salt tolerance on overexpression of *CaBiP1* in *Arabidopsis*. Herath et al. [81] identified three BiP in Solanum tuberosum and observed that one of the identified candidates, StBiP3, is induced by salt stress. CRT is a Ca2+ binding protein having molecular chaperon activity. Salt stress induces the transcript levels of CRT protein. Overexpression of CRT isoforms has been reported to increase stress tolerance in wheat [79] and tobacco [82].

ER stress signalling is also known to induce transcription of salt-responsive genes. Under salt stress, ABA levels increases which initiate proteolytic cleavage of an ER membrane-associated transcription factor bZIP17 by Golgi apparatus-resident site-1 protease (SIP). Processed bZIP17 is translocated to the nucleus to activate saltresponsive genes [83]. Liu et al. [44] showed that mutants of bZIP17 have increased sensitivity to salt stress. Ramakrishna et al. [84] reported increased salt tolerance in tobacco plant overexpressing bZIP17 from finger millet. Similarly, overexpression of ER-small heat shock protein (ER-sHSP), another ER-localised chaperon, has been reported to enhance salt tolerance in tomato (Fu et al., 2016). Guan et al. [85] characterised an ER-localised chaperon, namely Sensitive to Salt1 (SES1), which is induced by salt stress and activated by ER stress sensor bZIP17 by direct binding on the promoter region. These studies highlight the role of ER chaperones in stress signalling and acting as an important positive regulator of salt tolerance in plants.

In addition to chaperons, some enzymes involved in protein modifications also play an essential role in ERQC under salt stress. Blanco-Herrera et al. [86] observed that the mutants of UDP-glucose: glycoprotein Glucosyltransferase (UGGT), an enzyme involved in N-glycan protein modification of the misfolded proteins, are more sensitive to salt stress and negatively impacts plant growth.

Various ERAD components have been reported to act as both positive and negative regulators of salt tolerance in plants. In *Arabidopsis*, a mutant of ERAD components SEL1L/HRD3A and OS9 showed increased sensitivity to salt stress [87, 88], whereas Cui et al. [89] described a mutant for another ERAD component, ubiquitin-conjugating enzyme 32 (UBC32) to have enhanced salt tolerance in *Arabidopsis*.

Upregulation of UPR pathway genes has been associated with stress tolerance. Babitha et al. [90] reported that overexpression of a stress-responsive finger millet bZIP60 in tobacco leads to upregulation of UPR pathway genes such as BiP1, CRT1 and PDIL. The study proposed that the improved tolerance, in this case, could be through the UPR pathway.

As part of the salt stress signalling cascade, the components associated with the ERQC, ERAD and UPR mechanisms may work independently or may have regulatory connections with each other [45, 91–94]. Although many studies have reported the role of ER stress signalling in salt stress response, the underlying molecular

mechanism and client proteins for the ER-associated regulatory mechanisms are still unknown. Zhang et al. [95] performed the first ER proteome analysis of wheat seedlings to understand the role of ER proteostasis pathways in salt-induced growth reduction. They proposed a putative mechanism whereby salt stress generates ROS, which triggers Redox reactions in the cell leading to the accumulation of misfolded proteins. This increase in misfolded proteins in the ER lumen then triggers ER stress which further activates UPR to relieve ER stress and maintain proteostasis.

#### **7. ER stress signalling in response to cold stress**

Early perception of temperature fluctuation and remodelling of the cell membrane (or lipid bilayer) is the key to acclimation to sub-optimal growth conditions. Because ER is the site of fatty acid synthesis, changes in phospholipid composition in response to cold stress are expected to be determined in the ER. In this context, Tasseva et al. [96] investigated the ability of ER membranes to alter lipid composition in *Brassica napus* and reported desaturases involved in changing membrane fluidity. Cold stress leads to unstable membrane curvatures resulting from the accumulation of diacylglycerols (DAGs) [97]. Rupiz-Lopez et al. [98] revealed that two ER-localised synaptotagmin proteins, SYT1 and SYT3, remove DAGs to prevent PM damage caused by cold stress. Additionally, ER also hosts membrane proteins that act as a sensor of cold and help elicit a stress response. In this context, Ma et al. [99] identified CHILLING TOLERANCE DIVERGENCE 1 (COLD1), a plasma membrane (PM) and ER-localised protein in rice which triggers Ca2+ signalling in response to cold stress. Orthologs of COLD1 have also been identified in wheat [100] and Maize [101]. Overexpression of calcium-dependent protein kinases and calreticulin, also hosted by ER, has been reported to increase cold tolerance in rice [102]. William et al. [103] characterised a member of the Bcl-2-associated athanogene (BAG) family, AtBAG7 affecting cold tolerance in *Arabidopsis* is localised to the ER. Recently, Hu et al. [104] identified an ER-localised Sugars Will Eventually be Exported Transporters (SWEETs) which confers cold tolerance by regulating sugar metabolism and compartmentalisation under cold stress in *Arabidopsis*. Although many transporters situated in ER are involved in cold tolerance, our understanding of the underlying molecular mechanism is still limited.

#### **8. ER stress signalling in response to biotic factors**

Interactions of plants with various organisms play an essential role in its adaptability to the changing environment, not just in protecting it against various pathogens but also in enhancing its defence mechanisms [105–107]. When plants are subjected to microbial attack, pathogenesis-related (PR) proteins are synthesised as a response to it. A form of the immune response, Systemic Acquired Resistance (SAR), is established as a result of the NONEXPRESSOR OF PR GENE1 (NPR1)-regulated expression of PR genes in *Arabidopsis* [108]. Thus, it is conceivable that part of the plant immune response, including SAR, requires the synthesis and subsequent secretion of some PR proteins toward microbial pathogens. Consistent with this notion, the expression of several genes involved in protein secretion is induced in SAR of *Arabidopsis* in an NPR1- dependent manner, implying increased capability for SAR via transcriptional regulation that requires NPR1 function [109]. It was also reported

in tobacco (*N. tabacum*) plants that induction of the lumenal binding protein, an ER-resident chaperone, occurs rapidly during pathogen attack and precedes the expression of PR genes [110].

Several studies have demonstrated the involvement of ER bodies in plant defence mechanisms. Even if there is an artificial induction of a wound on a plant using the wound hormone jasmonic acid, which mimics the pest chewing damage, ER bodies are stimulated [111, 112]. In a study involving Brassicaceae plants, which are resistant to arbuscular mycorrhizal fungi (AMF) symbiosis, the plants were found to be susceptible to non-AMF groups (*Piriformospora indica* in particular), which in turn enhanced the growth of the plant [113] and showed that ER bodies act in the defence mechanism [114]. *Arabidopsis* mutants were impaired in the expression of PYK10, a

#### **Figure 1.**

*Summary of ER responses to biotic/abiotic stress conditions. Under biotic/abiotic stress conditions in plants such as virus infection, stress-related hormones and heat and osmotic stress, several factors including BiP, IRE1, bZIP60, bZIP17/28, genes involved in NPR1-depenedent pathways. Uncharacterized molecular pathways are indicated in dashed arrows. (adapted from [47]).*

target gene of *P. indica* in the roots of *Arabidopsis* [115]. PYK10 is a gene for an abundant myrosinase located in the ER [116, 117] which has spindle-shaped structures and is named ER bodies [27, 118, 119]. ER bodies can also be induced in rosette leaves by JA [120], and the jasmonate-insensitive coronatine insensitive1 [121] mutant does not form ER bodies [111].

#### **9. Conclusions**

Plants experience several stresses during their lifetime and in order to survive them, they should have a fast and dynamic response mechanism. Since nearly one third of the plant cell proteins responsible for being affected by stress are located in the ER, it has been studied that ER expresses differential responses in the plant in response to the situation. These dynamic responses in plants include changing the proteome by changing the gene expression patterns. It is the prime organelle that regulates the stress responses as any stress which affects protein folding leads to a cellular homeostatic response mediated by the UPR in response to ER stress. **Figure 1** highlights critical ER responses against perceived biotic/abiotic stress conditions.

### **Acknowledgements**

V.J.B. and B.S. thankfully acknowledge the Department of Biotechnology, Govt. of India for providing research grant to Assam Agricultural University and Dibrugarh University [Grant No. BT/PR25099/NER/95/1014/2017, dated 29 May 2018 and Grant no. BT/PR36255/NNT/28/1728/2020, dated 01/12/2021]. V.J.B. also acknowledge the support rendered by DBT e-Library Consortium (DeLCON) to Centre for Biotechnology and Bioinformatics at Dibrugarh University.

### **Conflict of interest**

The authors declare no conflict of interest.

*Responses of Endoplasmic Reticulum to Plant Stress DOI: http://dx.doi.org/10.5772/intechopen.106590*

#### **Author details**

Vishwa Jyoti Baruah1 \*, Bhaswati Sarmah2,3, Manny Saluja4,5 and Elizabeth H. Mahood3

1 Centre for Biotechnology and Bioinformatics, Faculty of Biological Sciences, Dibrugarh University, Dibrugarh, Assam, India

2 Department of Plant Breeding and Genetics, Assam Agricultural University, Jorhat, Assam, India

3 Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, USA

4 KWS Gateway Research Center, LLC (KWS), St. Louis, MO, USA

5 Boyce Thompson Institute, Ithaca, NY, USA

\*Address all correspondence to: vishwabaruah@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Updates on Endoplasmic Reticulum*

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Section 2
