Introductory Chapter: Biology and Biotechnological Applications of Chloroplasts

1. Introduction

Chloroplasts are semi-autonomous, photosynthetic organelles that play an essential role in photosynthesis. Beyond their basic role in photosynthesis, these extraordinary structures are critical to a variety of important cellular processes, including the response to stress and the synthesis of various biomolecules including vitamins, lipids, amino acids, phytohormones, and products of secondary metabolism [1]. Chloroplast transformation presents numerous benefits, such as the precise integration of a transgene at a predetermined location within the plastome through homologous recombination. It avoids issues such as gene silencing and position effects, ensuring elevated levels of transgene expression due to the high ploidy plastome. Furthermore, maternal inheritance prevents transgenic leakage through pollen, making transplastomic GMOs environmentally friendly. Numerous valuable traits have been purposefully incorporated to enhance the agronomic performance of crop plants. The chloroplast transformation system has proven instrumental in the expression of industrial enzymes and therapeutic proteins, indicating it as a promising solution to address persistent issues surrounding food security, drug manufacturing, and sustainable energy generation [2, 3]. Chloroplasts have firmly established their capabilities by proficiently expressing transgenes, and packaging recombinant proteins, thus protecting them from cellular degradations. This makes it easier to produce highly useful proteins on a large scale. Various segments of the chloroplast genome have served as stable markers for identification and evolutionary research. The ability of chloroplasts to produce tannin and chlorophyll also provides a sustainable pathway for the synthesis of a variety of natural pigments [4]. This boosts not only the market value of these plants but also endows them with biologically beneficial and environment-friendly attributes. Additionally, chloroplasts interact with the nucleus and other organelles of the cell to organize the acquisition of essential molecules needed for their resistance to stressful environmental conditions. They act as sensitive environmental sentinels that effectively produce substances to mitigate stress and, particularly, induce the activation of genes that are encoded by the nucleus, to develop stress resistance (Figure 1).

Figure 1.

Graphical representation of chloroplast biology and biotechnological interventions through plastome engineering.

2. Advances in chloroplast genomics

Plastid genomes, the ‘plastomes’ contain 120–130 genes and the majority of them are responsible for encoding critical parts of the organelle’s gene-expression machinery and substances necessary for photosynthesis. These genes are neatly arranged within nucleoids. Tobacco was the first higher plant whose plastid genome was sequenced, opening the door for the sequencing and characterization of hundreds of new higher plants. Researchers now have access to an extensive amount of plastid genome data that will help them better understand how the genes in these plastids are functionally characterized. Many of the genes discovered in chloroplasts have undergone in-depth analysis to see how they affect the stability and metabolic functions of these organelles. A collection of genes called hypothetical chloroplast open reading frames (ycfs) is still unknown to have all of its specialized roles fully understood. Some of these ycfs have been classified as non-essential, while others have been classified as essential elements [5].

Recent advancements in sequencing technologies, such as next-generation sequencing (NGS), have made it possible to quickly and affordably conduct complete chloroplast genome sequencing. This is because chloroplast genomes contain an enormous amount of valuable information that is useful for species identification, phylogenetic inferences, and population genetic studies. Chloroplast genome analysis is more effective in understanding the complexities of progeny evolution and their evolutionary relationships. This decision is based on the haploid structure, maternal inheritance, and unusually well-preserved genes [6]. Furthermore, in recent times, chloroplast genomics has emerged as a highly promising tool extensively employed in phylogenetic investigations, owing to its exclusive maternal inheritance and absence of recombination events [7]. The study of chloroplast genetics has played a pivotal role in unraveling the dynamics of gene distribution, cytoplasmic diversity, and population divergence. Recently, chloroplasts have been associated with a number of vital functions within numerous plant processes, including plant defense mechanisms, the synthesis of defensive compounds, as well as the regulation of plant physiology, development, and the alternative splicing of transcripts [8, 9].

3. Plastids as a source of natural and engineered products

Innovative advances in the synthesis of chlorophyll and tannin have led to the development of their liquid, paste, and dry forms. This improvement in the production of natural pigments raises their market value while also promoting biologically favorable qualities that are in line with their sustainability and health advantages. Chlorophyll and tannin are abundantly available in wood waste, especially from forestry operations that use mangrove trees. These organic substances have the potential to replace synthetic pigments, in sustainable ways which are frequently connected to environmental issues. Utilizing wood waste in this way could promote and provide business opportunities in forest villages, mangrove industry hubs, and small- to medium-sized batik and weaving businesses. Batik artists who use coastal and inland motifs seem to prefer using natural chlorophyll/tannin hues. Therefore, research into different natural sources of tannin that are safe for food and drink as well as the human body is crucial for fabric and batik coloring.

Over the past few years, advances in chloroplast engineering have shown great promise in addressing critical global challenges such as ensuring enough food, producing medicine, and generating sustainable energy for our growing world population. Chloroplasts have proven to be effective at expressing transgenes and protecting the recombinant proteins from cellular processes, resulting in highly functional proteins. This characteristic has also been useful in the field of RNA interference technology. In addition to the practical advantages of chloroplast transformation, such as the elimination of positional effects, the capacity for polycistronic expression, and significant protein production, this method indicates a development in biosafety; however, even if its great biotechnological potential, crops that have efficiently transformed are still a proof of concept. Despite rigorous efforts, a few important crops have shown to be resistant to chloroplast transformation, which limits their ability to be grown more widely. This chapter covers the most recent developments in this field as well as the challenges that still need to be overcome before this method can be applied to more crops and become an important tool in the field of plant biotechnology.

4. Plastid engineering for crop improvement

The incorporation of specific genes into the chloroplast genome has proven effective in enhancing important agricultural qualities in crop plants. Due to its numerous benefits when compared to nuclear transformation, plastid modification has emerged as a powerful method for the genetic engineering of significant crops. Potato [10] and sugarcane [11] are the most recent examples. Many initiatives to increase food yields concentrated on improving plants’ capacity for photosynthetic activity. Rubisco, the key enzyme at the center of photosynthesis, is made up of two parts: a large subunit encoded in chloroplasts and a tiny subunit encoded by the nucleus, which is later imported into the chloroplast. Similar to this, efforts have been made to change either the RuBisCO big subunit, the small subunit, or both parts [12] attempted to express complete RuBisCO protein in tobacco from Synechococcus elongatus and found CO₂ fixation rate and carboxylase activity of the RuBisCO to be increased. Another option for enhancing photosynthetic carbon fixation and agricultural yield is to increase the concentration of CO₂ in plastids. The introduction of the cyanobacterial bicarbonate transporter into the tobacco plastid genome failed to yield a significant enhancement in photosynthetic efficiency. However, when fructose-1, 6-sedoheptulose-1, and 7-bisphosphatase were expressed in both lettuce and tobacco chloroplasts, a notable boost in the productivity of these genetically modified plants became evident. The chlB gene from Pinus thunbergii, which is chloroplast-encoded, was also discovered to encourage tobacco root growth and early chlorophyll pigment development [13]. In order to increase biomass production, research is being done to convert C3 plants to C4 by modifying the RuBisCO large subunit and photo respirational pathway [14].

Since 1994, insect-resistant crops have flourished in fields, but Bt crops are raising concerns about the development of resistance. The development of insect-resistant transplastomic plants is a promising strategy, leveraging the benefits of transplastomic technology that have already been mentioned [10]. On the other hand, dsRNA expression, which targets an important insect gene in transplastomic plants, has been tested as a unique non-Bt-type insect resistance method. Within 5 days of feeding, RNA interference caused the target gene to be disrupted, killing all adult beetles and larvae [15]. When the agglutinin gene (pta) was expressed in leaf chloroplasts, aphids, lepidopteran insects, and bacterial and viral diseases were all successfully resisted [16]. The integration of the CeCPI gene (derived from sweet potatoes) and chitinase from Paecilomyces javanicus into tobacco plants conferred resistance not only to a wide range of pests and diseases but also rendered the plants resilient to salinity, osmotic stress, and oxidative challenges [17]. Proposing a potential defense against damage from excessive oxidation, increasing the activity of the mdar gene in tobacco plastids, and merging these chloroplasts with Petunia cells were suggested. This modification also seemed to make tobacco plants more resilient to oxidative stress. Additionally, when tobacco plants were genetically altered with the cyanobacterial flavodoxin (fld), they became better at handling oxidative stress. Furthermore, tobacco plants modified with the panD gene showed not only a 30–40% boost in their growth but also proved to be more resistant to high temperatures. Similarly, when arabitol dehydrogenase (ArDH) was introduced into tobacco chloroplasts, it allowed the plants to survive even in environments with extremely high salt concentrations, such as 400 mM NaCl [18]. This may open up novel opportunities for understanding how to cultivate resilient plants that can withstand stress and the value of transplastomic technologies.

5. Conclusion

In conclusion, chloroplasts are not only the most active cell organelle metabolically but also hold an immense potential for various biotechnological applications. Chloroplast being the main site of photosynthesis plays a pivotal role in a plant’s responsiveness and mitigation to biotic as well as abiotic stresses. Studying its basic biological attributes helps to unfold new possibilities of innovations in agriculture, biopharming, renewable energy, and beyond.