CHAPTER 2: THE ENHANCEMENT OF FUNDAMENTAL PHOTOSYNTHETIC PROPERTIES, GROWTH AND YIELD IN MR219 AND MR263 RICE VARIETIES VIA EARLY-STAGE CO2 ENRICHMENT TO THE SEEDLINGS BEFORE TRANSPLANTING (THESIS)

LITERATURE REVIEW

1.1 World and rice

Rice is one of the world's three most important food crops, including wheat and maize. Together, these three main cereal crops supply over 42% of all calories consumed by the entire population. In 2009, human consumption accounted for 78% of total rice production, compared to 64% for wheat and 14% for maize (GRiSP, 2013).

The current global population has reached 7.6 billion; the UN (2020) estimates it will reach 8.2 billion in 2025 then 11 billion in 2100 (Figure 2.1 A). As for Malaysia, the population is expected to reach 35 million in 2025 and 40 million in 2100 (Figure 2.1 B). For most Asian countries, rice is the staple food thus its consumption accounts for 80% of the world's total rice consumption (Sarena et al., 2019). Moreover, rice demand also grows in other regions such as Africa and Latin America, emphasizing its future market as the population grows (Muthayya et al., 2014).

Malaysia's milled rice consumption was 2.7 million metric tonnes in 2018 (Ministry of Agriculture and Agro-Based Industry Malaysia, 2019). According to Organisation for Economic Co-operation and Development & Food and Agriculture Organization (2020) reports estimates, the consumption trend was projected to increase as the domestic population grew. FAO (2020) trend analysis (Figure 2.2) shows that the national harvested rice area is almost constant annually. The gap between rice production and consumption in Malaysia is likely to widen, particularly when the yield per hectare rises slower than the consumption growth.

Figure 2.1 : Historic, current and projection of total (A) world and (B) Malaysia population from 1950 to 2100.  Adapted under Creative Commons license CC BY 3.0 IGO from World Population Prospect by United Nations DESA, Population Division, 2016

Retrieved from http://populatopm.un.org/wpp

Figure 2.2 : A trend in rice production area and yield harvested in Malaysia since 1994 to 2018 (FAO, 2020) 

Retrieved from http://www.fao.org/faostat/en/#country/131 

1.2 Rice botany 

Rice is now widely planted in over 100 countries worldwide, mainly in Asia's tropical and subtropical regions. It belongs to the tribe Oryzeae of the grass family Poaceae (formerly Gramineae). The genus is Oryza, consisting of two cultivated species, namely O. sativa and O. glaberrima and 22 other wild species distributed across the tropics and subtropics (Ge et al., 1999). Generally, O. sativa is referred to as Asian cultivated rice, while O. glaberrima is commonly known as African rice.

The shapes and texture of rice grains naturally classify themselves into two main subspecies: indica and japonica. In general, indica grains are long and slender in appearance and non-sticky when cooked. In contrast, japonica grains are short and round with a sticky texture when cooked. Moreover, indica and japonica rice also display significant differences in agronomics traits including plant height, leaf characteristics, plant types, and disease resistance (Vijay & Roy, 2013; Yang et al., 2014; Mahesh et al., 2016; Li et al., 2019). 

Indica varieties are grown generally in the submerged tropics and subtropics of south and southeast Asia. The indica stature is generally taller than the japonica subspecies with light green leaves with broad to narrow shape leaves. The grain's characteristics also tend to shatter easily and have a high amount of amylose content. In contrast, the japonica subspecies is mainly grown in temperate and colder zones such as the northern latitudes of East Asia. Japonica type also can be found in upland zones and high elevations in southern Asia. Japonica stature is relatively shorter than indica in terms of plant height and has less tiller number. The leaf shape is narrow and pointier with dark green leaves (Vijay & Roy, 2013). The grain is non-shattering with low amylose content. However, japonica has strong lodging resistance and cold tolerance (X. Wei & Huang, 2018).

1.3 The Rice Plant

1.3.1  General Growth and Development

The life cycle for each commercial rice variety is generally known and documented. This event depends largely on the growing environment, such as water availability and solar radiation distribution. Categorization of variety is based on the days it takes to reach maturity namely short-duration (less than 120 days), medium duration (120−140 days), and long duration (more than 160 days) varieties (IRRI, 2015a).

Rice has three main developmental phases (Parul, 2017) as follows:

      i.              Vegetative - start from seed germination to panicle initiation.

      ii.              Reproductive - from panicle initiation to flowering.

     iii.              Ripening - from flowering, grain filling to maturity.

The duration for each of these main phases greatly influences the number of panicles per area, average seed number per panicle as well as the average weight for the individual seed. These yield components determine the overall grain yield (Moldenhauer et al., 2018). These phases are subsequently divided into detailed growth developmental stages or periods (Figure 2.3).

Figure 2.3 : A typical life cycle duration for transplanted rice plants until harvest (IRRI, 2015a)

Retrieved from http://www.knowledgebank.irri.org/step-by-step-production/pre-planting/crop-calendar

1.3.2  Rice planting systems

In Malaysia, rice cultivation is a major agricultural activity, and several methods are used for planting rice, including direct broadcasting and rice transplanting methods. Here is an explanation of these methods according to Chan & Daud, (2016):

i. Direct broadcasting: In this method, the rice seeds are broadcast directly onto the prepared field. The field is flooded with water, and the seeds are allowed to germinate and grow. This method is commonly used in areas with low water availability and is less labour-intensive than transplanting.

ii.  Rice transplanting method: In this method, the rice seeds are sown in a nursery tray filled with a mixture of burned rice husk with or without topsoil. The seedlings are nurtured in the tray until they are around 15-20 days old and then transplanted into the main field. The field is prepared by ploughing and harrowing, and the rice seedlings will be transplanted using a transplanter machine. The seedlings are planted at the desired spacing and depth, and the field is flooded with water. This method is used to produce high-quality seedlings and to increase crop yield.

In Malaysia, direct broadcasting is commonly used in areas with low water availability, while rice transplanting is used to produce high-quality seedlings and increase crop yield. Both methods have their advantages and disadvantages and are suitable for different situations.

1.3.3              Vegetative Phase 

Rice vegetative phase length predominantly determines the overall growth duration of rice varieties. Some short-duration varieties have a relatively short vegetative phase, but some varieties have both shortened vegetative and reproductive phases. Regardless, rice undergoes the following processes during the vegetative phase:  

a. Germination stage: The vegetative process commences with seed germination. Rice seed germinated from the germinating embryo parts namely radicle and coleoptile

b. Seedling stage: Seedling develops seminal roots and is generally considered from germination to the fifth leaf growth. For transplanted seedlings, there is a recovery period covering the point from seedling uprooting to full recovery.

c. Tillering stage: This stage starts with the first tiller from the axillary bud and then increased rapidly (active tiller stage) until it reaches the maximum tillering stage. Tillering stopped after generating tertiary tillers.

d.  Inter-node elongation stage: The stage starts with internode elongation at the base of the culm (Figure 2.3)

1.3.4 Reproductive phase 

a. Panicle initiation (PI) to the booting stage: This phase is marked by the initiation of the panicle primordium of the microscopic dimension at the growth shoot level. Booting is the latter part of the panicle development stage. Approximately 16 days following visual panicle initiation, the sheath of the flag leaf swells. This swelling of the flag leaf sheath is called booting.

b. Heading stage: This stage happens as panicles gradually emerge out of the flag leaf sheaths. Emergence continues until 90-100% of the panicles are out of the sheaths.

c. Flowering stage: Flowering (blooming) or anthesis begins with the abundance of dehiscing anthers in the terminal spikelets on the panicle branches. Flowering continues successively until all spikelets in the panicles bloom. Pollination and fertilization then follow.

1.3.5 Ripening phase 

Rice grains develop upon pollination and fertilization and subsequently undergo distinct changes before fully mature. These stages are:

a.  Milk stage: Caryopsis contents are first watery then turn into milky inconsistency.

b. Dough stage: The milky caryopsis turn into a soft dough consistency which subsequently hardens

c. Maturity stage: The individual grain matures when caryopsis is fully developed in size, hard, clear, and free from greenish tint. This stage is complete when more than 90% of the grains are fully ripened.

1.3.6 MR 219

Rice variety MR 219 was developed and officially released by MARDI in 2001. It was the first variety designed for the direct seeding planting system (MARDI, 2000) and produced by the crossing between MR 137 and MR 151. The variety development was focused on panicle characteristics, mainly the number of grains/panicle and grain size (Table 2.1). Its high-yielding potential had made it a popular choice as a cultivated variety, accounting for over 90% of paddy cultivation area coverage in Malaysia for more than 20 years (40 planting seasons) (Hussain et al., 2012). The variety can produce more than 10 metric tonnes per hectare with proper water and fertilizer input management. It has a short maturation period, and tall but strong culms with disease resistance to blast and bacterial leaf blight.

1.3.7 MR 263

MR 263 also is a rice variety developed by MARDI and was released in 2010. The variety resulted from the crossing of two mutant parents, SPM 158 and MR 221 (MARDI, 2010). SPM 158 was developed from the induction mutation of a traditional variety namely Kurau Wangi, whereas the MR 221 results from the Q 31 induction mutation. The crossing between SPM 158 and MR 221 eventually resulted in MR 263 which had improved rice yield and resistance to main pests. MR 263 has a shorter plant height than MR 219, thus sturdier against lodging. It also has a shorter maturation period by eight days earlier than MR 219. (Table 2.1). Figure 2.4 provides a visual comparison between MR 219 and MR 263 at the plant (Figure 2.4A), panicle (Figure 2.4B) and spikelet-grain levels (Figure 2.4C)

Table 2.1 : MR219 and MR263 rice variety characteristics 

(MARDI, 2000 & 2010)

	MR 219	MR263
Released year	2001	2010
Maturity period (day)	105-112	97-104
Plant height (cm)	83-87	59-71
Panicle length (cm)	24.5	25
Seed (a)    Length (mm) (b)    Width (mm) (c)    1000 seed weight (g)	10.40 2.27 27.10	10.13 2.99 26.00
Pest/Disease resistance (a)    Leaf blast (b)    Panicle blast (c)    Bacterial Leaf Blight (d)    Brown Plant Hopper	Moderate resistant Susceptible Moderate resistant Moderate susceptible	Moderate resistant Susceptible Moderate susceptible Moderate resistant
Processing recovery (%) Head rice (%) Processed rice length (mm) Processed rice width (mm) Length width ratio Amylose content (%)	65.00 72.00 6.90 2.10 3.29 20.10	67.20 82.90 6.67 2.16 3.13 19.97

Figure 2.4 : Representative of (A) MR219 and MR263 plant appearance, (B) MR219 and MR263 panicles and (C) MR219 and MR263 spikelets and grains (Hussain et al., 2012)

1.4 Rice Stomata

Stomata are pores on the leaf formed by a pair of guard cells whose movement through the change in turgor pressure controls the pore aperture (Negi et al., 2013). Stomata on plants can occur in a number of locations leading to leaves that are amphistomatous (stomata on both sides), hyperstomtous (stomata on the upper surface) or hypostomatous (stomata on the underside) (Parkhurst, 1978). An isostomatous leaf has stomata that occur with approximately equal frequencies on both surfaces. Stomata create gateways to optimize carbon uptake for photosynthesis while minimizing excessive water loss from transpiration by modulating stomatal conductance (g_s) in response to environmental stimuli (Farquhar et al., 1980; Kim et al., 2010).

Morphologically stomata are classified according to the shape of the guard cells. In dicots (e.g., Arabidopsis) they are kidney-shaped while in monocots such as rice in this study, they are dumbbell-shaped. Despite the distinct physical features, both types of stomata generally develop in a similar fashion involving sequential asymmetric and symmetric cell division of specialized epidermal cells (Bergmann & Sack, 2007; Buckley et al., 2020).

Fundamentally, the destined epidermal or protodermal cell divides asymmetrically to produce a meristemoid (transient cell state of the stomatal lineage) which differentiates into a guard mother cell (GMC). This cell then eventually divides symmetrically to form a pair of guard cells forming a unit of a stoma. Even though both stomata types, in general, follow most of this developmental course, in detail they do differ particularly in the final stages wherein rice the GMC induces asymmetrical divisions into subsidiary mother cells (SMC) that finally become two subsidiary cells (SCs) at the opposite ends of GMC, as well another one symmetric division forming a pair of guard cells. Thereby, stomata in rice come in a complex form of four cells (a pair of guard cells being flanked in between two subsidiary cells) (Figure 2.5). Moreover, it is worth noting that in Arabidopsis the stomata are scattered across the leaf without clear developmental zonation while in rice stomata occur orderly in specific cell rows on both leaf surfaces (Wei et al., 2020).  

Figure 2.5 : A typical sequential event of stomatal complex formation in rice (GMC, guard mother cells; SMC, subsidiary mother cells) 

Adapted from Wu et al., 2019

1.5 Photosynthesis

Photosynthesis is a collection of biochemical processes on how plants convert Carbon dioxide (CO₂) and light to glucose (C₆H₁₂O₆) and generate Oxygen (O₂). Photosynthesis can be summarised by the word equation as below (Figure 2.6):

Figure 2.6 : Chemical equation of photosynthesis (Kirkpatrick, 2018) 

Retrieved from 31 December 2021 from  https://www.researchgate.net/figure/The-chemical-equation-of-photosynthesis_fig1_327920538

The process of photosynthesis as a whole in the plant is based on two reactions that are carried out in separate locations of the chloroplast namely the light reaction and the Calvin cycle (Johnson, 2016). Although these two reactions occur at different locations in the chloroplast, these reactions are closely linked and each of the reactions has sophisticatedly played a significant role in the photosynthesis process. Undoubtedly, photosynthesis is an essential process that occurs in the plant as it is fundamental to plant growth and the key determinant of crop yield (Evans, 2013). The efficiency by which a crop absorbs light energy and converts it into plant biomass throughout the growing period is a primary determinant of final yield (biomass or grain) (Long et al., 2006; Simkin et al., 2019). However, photosynthesis is influenced by a range of environmental factors such as CO₂ concentrations, light, temperature, and as well as indirect influences such as relative humidity and soil moisture (Yaapar, 2017). Among these factors, CO₂ concentrations and light are the major limiting factor of photosynthesis (Sarkar, 2020). Hence, the dependence of photosynthesis on environmental factors is very important to farmers and agronomists as crop productivity is highly dependent on the general rate of photosynthesis in an ever-changing environment.

1.5.1  Light Reaction

As mentioned above in section 2.5, photosynthesis is based on two reactions that are carried out in separate locations in the chloroplast (Figure 2.7 A). The light reaction takes place in the thylakoid membrane (Figure 2.7 B). The final goal of the light reactions is to convert light energy into chemical energy and this chemical energy, particularly Adenosine triphosphate (ATP) and Nicotinamide adenine dinucleotide phosphate (NADPH) produced will be used in the next reaction, which is the Calvin cycle to fuel the assembly of sugar molecules (Alberts et al., 2002).

(Nature Education, 2014), Retrieved 31 December 2021 from www.nature.com/scitable/content/structure-of-a-chloroplast-14705175/. (B) shows the process of light reaction occurring in the thylakoid membrane. Adapted under Creative Commons license CC BY 3.0 from The Light-Dependent Reactions of Photosynthesis by OpenStax College, Retrieved 31 December 2021 from http://cnx.org/contents/f829b3bd-472d-4885-a0a4-6fea3252e2b2@11. (C) shows the Calvin cycle reaction takes place in the stroma. Adapted under Creative Commons license CC BY 3.0 from The Calvin Cycle by OpenStax College, Retrieved 31 December 2021 from http://cnx.org/contents/b3c1e1d2-839c-42b0-a314-e119a8aafbdd@9.10

The light reaction begins in a grouping of pigment molecules and proteins called a photosystem. Photosystem is a grouping complex of proteins and pigments (light-absorbing molecules) that are optimized to harvest light (Yahia, 2018). There are two types of photosystems namely photosystem I (PSI) and photosystem II (PSII). Both photosystems contain numerous pigments to harvest light energy, as well as a special pair of chlorophyll molecules at the reaction centre of the photosystem.

When light energy is absorbed by the light-harvesting pigment pigments in PSII, the energy is transferred to P680, boosting an electron to a high energy level. This high-energy electron then travels down an electron transport chain (ETC) and loses some energy as it goes (Leegood, 2013). The energy is used to pump the Hydrogen ions (H⁺) from the stroma into the thylakoid interior which in turn will result in building a concentration gradient in the thylakoid interior. As the H⁺ ions flow down the concentration gradient and back into the stroma, they pass through ATP synthase and drive ATP production (Demirel, 2014).

On the other hand, in PSI, the electron joins the P700 special pair of chlorophylls in the reaction centre and again is boosted to a very high energy level. This high energy level of the electron is transferred to an acceptor molecule and travels down the second leg of ETC. At the end of the chain, the electron is passed to NADP⁺ to produce NADPH.

1.5.2 Calvin Cycle

The light-independent reaction, also known as the Calvin cycle utilises the products from the light reactions, ATP and NADPH to produce sugar molecules which are essential for plant growth and development (Raines, 2003). Unlike the light reaction which takes place in the thylakoid membrane, the Calvin cycle reaction takes place in the stroma (Figure 2.7 C). This cycle involves 11 different enzymes, which catalyses 13 reactions, and is initiated by the enzyme ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) (Raines, 2003). Calvin cycle is partitioned into 3 phases namely the fixation phase, reduction phase and regeneration phase (Heineke & Scheibe, 2009).

In the fixation phase, when carbon dioxide (CO₂) enters the interior of a leaf via stomata and diffuses into the stroma of the chloroplast, the carbon atom is fixed into a five-carbon acceptor molecule, ribulose-1,5-bisphosphate (RuBP) and a six-carbon compound. This six-carbon compound is extremely unstable and splits into two molecules of a three-carbon compound, 3-phosphoglyceric acid (3-PGA) and this reaction are catalysed by the enzyme rubisco (Senatore et al., 2020). In the reduction phase, 3-PGA molecules are converted and reduced into a three-carbon sugar called, glyceraldehyde-3-phosphate (G3P) by consuming ATP and NADPH from the light reaction. The regenerative phase of the cycle involves a series of reactions that convert G3P into the CO₂ acceptor molecule, RuBP. The majority of G3P produced in the reduction phase stays within the cycle. However, some G3P exit the cycle and are used to synthesise sucrose and starch.  For one G3P to exit the cycle (to synthesise sugar), three CO₂ molecules must enter the cycle allowing three new carbon atoms to be fixed to produce six G3P molecules. Only one G3P molecule can exit the cycle to a synthesis sugar molecule, while the other five must be regenerated into a RuBP acceptor (Raines, 2003).

The carbon fixation phase of the Calvin cycle is not truly perfect. Since Rubisco can have both carboxylase and oxygenase activities, there is a tendency for Rubisco to bind with oxygen (O₂) instead of CO₂ under certain conditions such as high temperature, drought and low CO₂/high O₂ ratio (Cheng-Jiang Ruan et al., 2012). This will lead to the unwanted process named photorespiration, which reduces the photosynthesis, particularly the Calvin cycle efficiency. Thus, numerous researches have been attempted and intensely taken place to minimize the process of photorespiration in the plant.

1.6 Approaches to evaluating the rate of photosynthesis 

The rate of photosynthesis is one of the key determinants of the final yield (seeds or grains). Numerous studies have been conducted to evaluate photosynthesis with the ultimate goal of improving plant growth performance and development. Light response curve and assimilation versus intercellular CO₂ response curve (A/Ci curve) are the common approaches used to study the photosynthesis rate.

1.6.1 Light response curve

The light response curve (LRC) describes the relationship between the rate of photosynthesis and a range of relevant light intensities (Herrmann et al., 2020). LRC is fitted by photosynthetically active radiation (PAR) against the rate of photosynthesis (μmol CO₂ m^-2 s^-1) (Figure 2.8). and the curves can be constructed by light response curve software or equipment such as the Licor portable photosynthesis machine. LRC provides information on the maximum photosynthetic capacity, quantum yield, dark respiration, light compensation point and light saturation point as well as leaf radiation use efficiency (Herrmann et al., 2020).

Based on Figure 2.8, photosynthetic assimilation is absent in the dark, and the CO₂ emitted is a result of mitochondrial respiration or dark respiration. As the PAR increases, the CO₂ uptake increases until it equals the CO₂ release to respiration (Taiz & Zeiger, 2010). At this point of PAR value, the net CO₂ exchange of the leaf is balanced (zero), and an increase above it results in a proportional increase in the rate of photosynthesis, which is known as the light compensation point (Valentine et al., 2013). The initial part is essentially linear, and its slope corresponds to the light-use efficiency by chloroplasts or photosynthetic efficiency (Ф). At a high PAR value, the photosynthetic response begins to plateau and hits its maximum capacity (maximum photosynthesis). Beyond this point, the rate of photosynthesis is unaffected by the rise of PAR value (light saturation point) (Lobo et al., 2013). Plants acclimated to high light commonly have a high light compensation point, light saturation point, as well as maximal photosynthetic rate generally (Huang et al., 2021). Hence, LRC can be used to identify CO₂ assimilation, photochemistry, photoacclimation, photoinhibition, and photoprotective mechanisms in different light conditions (Huang et al., 2021).

Figure 2.8 : A typical light response curve (Rivera-Méndez et al., 2017)

Retrieved 1 January 2022 from   www.scielo.org.co/scielo.php?script=sci_arttext&pid=S012099652017000300323

1.6.2              Assimilation versus intercellular CO₂ response curve (A/Ci curve)

A/Ci curve is a plot of photosynthetic CO₂ assimilation (A) versus CO₂ at intercellular space (Ci) or inside the leaf (Figure 2.9). The A/Ci curve describes how photosynthesis reacts to changing environmental conditions (Slot & Winter, 2017), which is a useful estimation to predict the plant’s capacity for carbon uptake in future climatic conditions (Lombardozzi et al., 2018). This curve can be constructed by the A/Ci Curve Fitting software or equipment such as Licor portable photosynthesis machine.

According to Stinziano et al. (2019), parameters derived from the A/Ci curve to calculate the biochemical limitation of photosynthesis include the maximum carboxylation rate of Rubisco (Vc_max), maximum rate of electron transport for the given light intensity (J_max) and maximum rate of triose phosphate utilization (TPU). Interestingly, other parameters could be obtained to estimate photosynthetic traits including dark respiration (R_d), and CO₂ compensation point (Γ), which can provide information on the balance between the rates of photosynthesis (Stinziano et al., 2019).

Figure 2.9 : Curve fitting for A/Ci, curves lettuce plants under red LEDs using the method developed by Sharkey et al. (2007). Blue dots represent averaged assimilation (µmol/m²/s) measured by CIRAS-3. Red lines represent the limitation imposed by Rubisco carboxylation activity when the CO₂ supply was low. Green lines represent the limitation imposed by electron transport rates as CO₂ concentration increases. Yellow lines represent limitations imposed by the rate of triose phosphate utilization, e.g the formation rate of the end-product of the Calvin cycle. Adapted from Light Spectrum Affects Maximum Rate of Carboxylation & Electron Transport by PP System

Retrieved 1 January 2022 from https://ppsystems.com/wp-content/uploads/AN_CIRAS-3_Light-Spectrum-Carboxylation-Electron-Transport.pdf

1.7 CO₂ enrichment

CO₂ enrichment, also known as CO₂ fertilization, refers to the practice of increasing the concentration of carbon dioxide in the air surrounding plants. This technique is often used in greenhouses and indoor growing environments to enhance plant growth and productivity (Sakai et al., 2019; Usui et al., 2016). Carbon dioxide is an essential component of photosynthesis, the process by which plants convert light energy into chemical energy to fuel their growth and development. Increasing the concentration of carbon dioxide in the air can stimulate photosynthesis and enhance plant growth, particularly for C3 plants, which include most crops and vegetables (Tomimatsu & Tang, 2016). Studies have shown that CO₂ enrichment can increase plant growth and yield by up to 40% in some cases. However, the benefits of CO₂ enrichment are highly dependent on the specific plant species, growing conditions, and the duration and intensity of exposure (Hu et al., 2022).

1.7.1 Stomatal response to CO₂ enrichment

Gas exchange between plants and the atmosphere is regulated by stomata. Environmental elements including water status, temperature, CO₂ level, and light may have an impact on stomatal behaviour. According to species and genotypes, stomatal short-term actions (such as stomatal closure) and long-term developmental (such as stomatal size and density) reactions to environmental changes may coexist (Gray et al., 2000; Hubbart et al., 2013). Stomatal conductance, stomatal density, and leaf transpiration rate are often decreased in response to elevated CO₂ (Xu et al., 2016).

Studies have suggested that elevated CO₂ can lead to a decrease in stomatal density in some C3 plants, including rice (Uprety et al., 2002). This reduction in stomatal density can lead to a decrease in transpiration rates, an increase in water-use efficiency, and a decrease in plant vulnerability to drought stress. Elevated CO₂ has also been shown to increase the size of stomata in some C3 plants, including rice. This increase in stomatal size may lead to a higher rate of CO₂ diffusion into the leaf and an increase in photosynthesis (Bertolino et al., 2019; Haworth et al., 2021; Woodward & Kelly, 1995).

Some studies have suggested that there may be a trade-off between stomatal density and size in C3 plants, with elevated CO₂ leading to a decrease in stomatal density and an increase in stomatal size (Driscoll et al., 2006; Uprety et al., 2002). This trade-off may help to maintain a balance between water-use efficiency and photosynthesis. As with the effects of stomatal conductance on elevated CO₂, the effects of stomatal density and size can also interact with other environmental factors, such as temperature, light, and soil moisture. These interactions can influence the overall response of C3 plants to elevated CO₂ (Xu et al., 2016).

1.7.2  Effects of CO₂ enrichment on crop photosynthesis

Plant responses to atmospheric carbon dioxide will be of great concern in the future, as the CO₂ concentration is predicted to rise continuously. To a certain degree, CO₂ enrichment or elevated CO₂ concentration leads to an increase in photosynthesis in plants, which in turn results in greater production of sugar molecules and biomass or even yield. Under CO₂-enriched conditions, increased photosynthesis is mainly due to the increase in Rubisco carboxylation activity. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme protein that catalyzes the first step in the Calvin cycle, carbon fixation and also the photorespiratory carbon oxidation (Spreitzer & Salvucci, 2002). As the name suggested (Ribulose-1,5-bisphosphate carboxylase/oxygenase), Rubisco can have both carboxylase and oxygenase activities (Kaul Wattal et al., 2015). The carboxylase reaction of Rubisco fixes carbon molecules to ribulose-1,5-bisphosphate (RuBP), a five-carbon compound and generates two molecules of 3-phosphoglyceric acid (3-PGA) (Wostrikoff & Stern, 2009), which 3-PGA will be further reduced to synthesize sugar molecule for the plant growth and development.

However, there is a tendency for Rubisco to bind with oxygen (O₂) instead of CO₂ (Oxygenase activity of Rubisco) under certain conditions especially low CO₂/high O₂ ratio, and high temperature (Cheng-Jiang Ruan et al., 2012). The Oxygenase reaction of Rubisco will lead to a process called photorespiration, an undesired process that is wasteful in terms of energy as this process costs the plant more energy and does not result in any gains of energy or carbon (Peterhansel et al., 2010). Moreover, Rubisco displays a significant affinity for O₂ and this characteristic has further diminished the overall efficiency of photosynthesis (Pribil & Leister, 2017). The property of Rubisco carboxylase activity or oxygenase activity is greatly regulated by the relative CO₂ and O₂ ratio in the atmosphere (Kaul Wattal et al., 2015). Hence, the CO₂ enrichment condition caused a higher ratio of CO₂ to O₂, which favours the carboxylation activity of Rubisco (Thompson et al., 2017) and at the same time reduces the occurrence of photorespiration, thereby increasing photosynthesis and carbon gain (Badger & Price, 2003). Under conditions of elevated CO₂, a decrease in stomatal conductance (gs) may restrict the rate of CO₂ fixation while promoting water use efficiency (WUE) to aid plant growth (Leakey et al., 2009).

A meta-analysis done by Hu et al.(2022) and Ainsworth & Rogers (2007) reported that elevated CO₂ increases the rate of photosynthesis in many C3 plants. This is due to the increased availability of CO₂ for the carboxylation reaction in the Calvin cycle. The extent of the increase varies among species and depends on other environmental factors such as light intensity, temperature, and nutrient availability. Elevated CO₂ generally decreases stomatal conductance in many plants. This is because plants need less stomatal opening to take up the same amount of CO₂ when the atmospheric concentration is elevated. Reduced gs leads to reduced transpiration, which can increase water-use efficiency and reduce water loss. Figure 2.10 illustrates how plant development under eCO₂ conditions on photosynthesis and stomatal conductance.

Figure 2.10 : Effects of eCO₂ on photosynthesis and stomatal conductance on plant growth responses. (Green circle = chloroplast; orange squares = epidermal cells; half circles = stomata; red arrow denotes an increase; and blue arrow denotes a decrease) (Gamage et al., 2018)

1.7.3 Effects of CO₂ enrichment on plant growth

Numerous studies have been actively conducted to evaluate the effects of elevated CO₂ concentration on C3 plants such as rice. Free Air CO₂ Enrichment (FACE) experiment by Ainsworth et al. (2008), results showed that photosynthesis in trees and shrubs to some extent in C3 plants has increased under CO₂ enriched conditions, leading to an increase in dry matter accumulation, leaf area and plant height. Abzar et al. (2017) reported considerably greater total rice plant biomass seedlings cultivated in eCO₂ settings within the glasshouse. Ainsworth & Long (2005) found that crop species, growing seasons, and experimental conditions all affected the amount of growth and above-ground biomass produced when exposed to conditions of rising CO₂ levels. An experiment conducted using FACE facilities displayed that a variety of crops and ecosystems exposed to high CO₂ have shown a range of increases in shoot biomass. At adequate nitrogen and water levels, C3 grass crops such as wheat, ryegrass, rice, and barley experienced average increases of roughly 17% and greater increases in shoot biomass (approximately 25%) in C3 legumes such as clover and soybean (Kimball, 2016). According to Masle (2000), eCO₂ affected and stimulated changes in wheat plant cell division, growth, and patterning. The presence of a high carbon supply in the environment has been investigated as a factor in meristematic tissues' expansion and rapid cell division, as well as in plants' improved initial growth and productivity (Thilakarathne et al., 2015). Figure 2.11 shows the mechanism of plant growth at eCO₂ which can lead to changes in carbon and nitrogen metabolism, changes in cell cycle properties, and hormone metabolism due to increased carbon supply to shoots and roots.

Liu et al. (2017) reported that the entire growth period for rice was shortened by 22.1% as compared to the control (ambient CO₂ concentration) under elevated CO₂ concentration (650 μmol·mol⁻¹) and temperature (28-33^oC). Most crop species exhibit accelerated flowering in response to elevated CO₂ (eCO₂), while some like sorghum show a delay. However, for soybean and maize, the flowering time varied- ranging from early, delayed or no change. The alterations in flowering time under eCO₂ may depend on other environmental factors like temperature and photoperiod. Species exhibiting early flowering under eCO₂ have less respiratory costs, while late flowering varieties often have lower respiratory rates (Springer & Ward, 2007).

Figure 2.11 : Effect of increased carbon supply at elevated [CO2] on other cellular processes and plant growth responses. (C = Carbon; N = Nitrogen; NO3− = Nitrate; red arrow denotes an increase; and blue arrow denotes a decrease) (Gamage et al., 2018)

1.7.4              Effects of CO₂ enrichment on yield component

The yield of rice and soybean under an elevated CO₂ environment (200 ppm above ambient) in the FACE experiment was 15% and 13% higher than the yields of crops grown in an ambient CO₂ environment (Han Yong Kim et al., 2003; Morgan et al., 2005). Rice grown under the traditional flooding method at elevated CO₂ concentrations ranging from 450–500 μmol mol⁻¹, resulted in an increase in spikelet number per panicle and filled spikelet, which led to an increase in rice yield by 25% (Yuting Li et al., 2017). In addition, the 1000-grain weight and harvest index (yield mass/above-ground vegetative mass)  of rice grown under the traditional flooding method and non-flooded film mulching method was increased when CO₂ concentration was elevated to 550 - 600 μmol mol⁻¹ (Yuting Li et al., 2017). Nevertheless, elevated CO₂ (200ppm above ambient) increased days to the maximum tiller number stage by approximately 4%, resulting in a greater number of tillers in rice (Usui et al., 2016).

A study by Bishop et al.(2015) examines the yield response of 18 different genotypes of soybeans grown under free-air CO₂ enrichment conditions. The results suggest that soybean plants are likely to experience an increase in yield in response to higher CO₂ levels. However, the degree of increase varies depending on the genotype of the soybean plant, and other environmental factors such as temperature and soil moisture also play a role in determining the plant's response to increased CO₂ levels. An increase in total seed mass and seed quantity was observed in 79 crop and wild species cultivated under eCO₂, according to a meta-analysis done by Jablonski et al.(2002). However, as the total plant biomass also grows, the harvest index is unaffected by an increase in the number of seeds (Bunce, 2017; Jablonski et al., 2002).

Chaturvedi et al. (2017) mentioned that under the eCO₂ condition, an increase in the activity of invertases and sucrose synthase has been noticed in rice grain development. These enzymes change sucrose into the monomeric hexoses needed to create starch. Because of this, the activation of these enzymes may result in an increased sink and more photoassimilates being transported to the grains. Most crop plants are sink rather than source-constrained, therefore increasing photosynthesis under eCO₂ and strengthening the sink may result in higher grain/seed output, as seen in rice (Nakano et al., 2017).

Table 2.2 : Example of the effects of elevated CO₂ on photosynthesis, biomass production, and grain yield in other C3 crops (Singer et al., 2020)

Crop	Method exposure	Photosynthetic rate	Biomass	Yield
Soybean Glycine max	FACE	↑17-26% in assimilation rate across varieties	↑ 13-32% biomass across varieties	↑0-20% across varieties
	Open top chamber in pots	No Data	↑ 4-53%	3-95%
	FACE	No Data	↑15-17% depending on the year	↑15-16% across years
	Open top chamber in the field	No Data	↑61% dry weight	No change
	Open top chamber in pots	↑27-46% in assimilation rate	↑ 15% in dry weight	↑13% across years
Field pea Pisum sativum	FACE	No Data	↑ 19-33% dry weight across varieties	↑26% across varieties
Mungbean Vigna radiata	FACE	↑ 7-19% in assimilation rate	↑11% in total dry weight	↑14%
Faba bean Vicia faba	Crop enclosure in the field	↑46% in assimilation rate	↑58% dry weight	↑51%
Lenti Lens culinaris	FACE	↑29-55% in assimilation rate depends on varieties	↑31-41% in dry weight and no change in root dry weight	↑55% across varieties
Chickpea Cicer arietinum	Open top chamber in the field	No Data	↑ in leaf dry weight and stem	↑10-13%
	Open top chamber in the field	No data	↑48-142% in stem dry weight and 50% flowering	No data

Note: FACE: Free-air CO₂ enrichment

1.8  Early-Stage CO₂ enrichment

Lake et al. (2002) reported that during the early stage of plant development, the structure of the leaf is sensitive to environmental factors, including CO₂ atmospheric level. The eCO₂ atmospheric conditions are detected by the mature leaves and transmit a long-distance signal mechanism that responds to young leaves' development (Lake et al., 2001). This CO₂ detection mechanism and signalling could enhance or optimize plant performance may be due to biochemical adaptations of photosynthesis and also a leaf structural response.

Early-stage CO₂ enrichment or CO₂ priming is a technique that involves exposing crops to elevated levels of carbon dioxide (CO₂) for a short period during their early growth stages. A study by Jitla et al.(1997) concluded that exposing rice seedlings to eCO₂ at the early shoot apex development is essential for obtaining maximum increases in grain yield. An increased supply of carbohydrates during the vegetative stage speeds up the pace of leaf development on the main shoot and each tiller, encouraging the emergence of new tillers. Some of the tiller buds stay permanently suppressed if high CO₂ exposure is postponed. Since the majority of tillers are capable of producing grain, the highest grain production increases under elevated ambient CO₂ concentrations can only be attained by subjecting plants to high CO₂ shortly after germination.

The significance of elevated CO₂ in promoting early leaf growth in grasses was similarly illustrated by Robertson et al. (1995) in wheat plants that were 7 days old. As early as 12 h post-mitosis, CO₂ enrichment significantly increased mitochondrial biogenesis in the cells of the developing zone at the base of the first leaf. At 650 ppm compared to 350 ppm, mesophyll cell and chloroplast volumes increased by 10 and 25%, respectively, showing that this early increase in cellular activity was translated into alterations in the mature leaf (Robertson et al., 1995; Robertson & Leech, 1995).