Full Thesis is on Research Gate DOI:10.13140/RG.2.2.14708.63368
CHAPTER 1: INTRODUCTION
1.0 Overview
In the first part of the introduction details of global rice production and the need to increase its production will be discussed before summarising what is known about the environmental regulation of leaf development, with specific emphasis on rice and the differentiation of stomata (small pores on the leaf surface with a key role in gas exchange). Primary food for human consumption comes from a limited number of cultivated grasses (rice, wheat, barley) of which rice is globally the most important. However, as global population increases and pressure on agricultural land and resources increases, there is an urgent need to improve grain yield while at the same time using available resources more efficiently, most notably land and water.
The source of all food is the carbon fixed from the atmosphere via photosynthesis. There is an extensive literature on the process of photosynthesis and numerous investigations have explored the possibility of improving the efficiency of this biochemical process and thus theoretically increasing yield (Andrews and Whitney, 2003; Whitney et al., 2011). However, to date this approach has met with little success. An alternative approach to improving the efficiency of photosynthesis is to consider the multicellular organ which has evolved to perform this process- the leaf. A leaf is an organ whose overall form and constituent cells enable the uptake of CO2 and water and the absorption of light energy so that photosynthesis occurs. The leaf must also facilitate the transport of the products of photosynthesis to other parts of the plant for growth and storage (Evans, 1975; Yoshida, 1972).
A significant body of evidence indicates that the overall form of a leaf and the differentiation of the constituent cells are subject to both an endogenous developmental regulation and influence by external environmental factors. In particular, light quality and intensity are known to influence leaf development with a direct outcome on the photosynthetic capability of the leaf (Pallardy, 2008). Most notably, stomata form and frequency have been identified as potential parameters for optimization which might be exploited to improve crop yield (Schluter et al., 2003; Franks and Beerling, 2009).
1.1 Rice and people
Rice (Oryza sativa L.) is touted as being fundamental to life owing to its prominent roles in shaping histories, cultures, diets and economics for half of humanity (Gomez, 2001). Today, rice feeds more than three billion people and more than one billion depend on rice cultivation for their livelihood (Skinner, 2012). Furthermore, it has supported a greater number of people for a very long period of time compared to any other crop since it was domesticated between 8,000 to 10,000 years ago (Greenland, 1997). Unlike maize or wheat, less than five percent of total rice production is traded on world markets, mainly within Asia and from Asia to Africa and Europe. For many Asian countries, rice self-sufficiency and political stability are interdependent issues (Fairhurst and Dobermann, 2002). In Asia two rice sub-species are widely cultivated namely indica and japonica and they have clear variation in the sequences of genome, physical and physiological properties such as hardiness and yield potential (Oka, 1958; Garris et al., 2005; Huang, X. et al., 2010)
1.1.1 Insufficient rice production
Together with wheat and maize, rice is regarded as one of the ‘Big Three Crops’ that feed the world. In fact, as a cereal grain, rice is grown on 6 continents and in more than 100 countries. Therefore, it is the staple food for more than half of the world population (more than 3 billion people globally, (IRRRI, 2015)). In terms of human consumption, especially in supplying calories, rice is by far the most important (Fig. 1.1A). Despite this, rice production is not the highest globally among the three crops mentioned (Fig. 1.1B). This worrying fact is indeed a problem as world population is increasing exponentially and over the next 40 years in Asia alone the population will increase by about 1.5 billion, more than a third of the present population (DESA, 2004).
Figure 1.1: World calories supply and production of the big three crops namely rice, wheat and maize in 2009. Summarized data for (A) obtained at: FAO, Accessed 16 November 2012. http://faostat.fao.org/site/609/default.aspx#ancor
Summarized data for (B) obtained at: FAO, Accessed 16 November 2012. http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor
Despite the modern age that we are living in now, about 854 million people are hungry and each day about 25,000 people die from hunger-related causes. Sixty percent of the world’s population lives in Asia, where each hectare of land used for rice production currently provides food for 27 people, but by 2050 that land will have to support at least 43 people (Sheehy et al., 2008). Looking at a global picture, world population is expected to grow by over a third, or 2.3 billion people, between 2009 and 2050. Nearly all of this growth is forecast to take place in the developing countries. Meanwhile, urbanization is expected to progress at an accelerating rate with urban areas to account for 70 % of world population in 2050 (49 % increment from present time) and rural population, after peaking sometime in the next decade, is actually declining (FAO, 2009).
By looking at the rice production graph in Fig. 1.2 A, in the last 50 years until recently (2013), rice production has steadily kept pace with the population growth rate, mainly due to the gains from the technologies of the green revolution era such as semi-dwarf and fertilizer responsive varieties, as well as other associated agronomic technologies.
Figure 1.2: Trends in rice production in Asia and globally (A) and regional population growth and projection (B). Both figures are adapted from Elert (2014).
However about a decade ago, rice yield growth had not increased tremendously compared to what had been obtained before the new millennium. In contrast, the same trend is not seen in the population growth (Fig. 1.2 B). World population constantly grows and is expected to further increase by over a third, or about 2.3 billion people, between 2009 and 2050. This imbalance between food supply and population growth has triggered concern globally and many endeavours are being attempted, such as precision farming and international trade in order to prevent havoc in the future. Therefore, increasing and improving rice production is indeed one of the important efforts that must be worked on for the sake of human sustenance.
1.2 Improved photosynthesis: An approach to enhance rice yield
Photosynthesis is the fundamental process in which plants synthesize carbohydrate and generate oxygen from carbon dioxide (CO2) and water by using light energy. Carbohydrate acts as a store of energy that serves as a resource to power cellular processes for all forms of life and to provide the carbon skeletons for a vast array of cellular products. However, photosynthesis is not a perfect process. It is the product of an evolutionary path which has set various constraints on its efficiency. Moreover, although people use plants as a source of food and energy (mainly via the investment of the products of photosynthesis into seed development), plant development is not primarily evolved toward a theoretical maximum to enhance seed yield (Gust et al., 2008). Due to these various constraints, reflecting both basic biochemical inefficiency in the process of CO2 fixation and the conflicting source and sink relationships within a plant, the efficiency of solar energy conversion into biomass for most crops typically does not exceed 1% (Zhu et al., 2010; Walker, 2009). As a consequence, improving the efficiency of photosynthesis has been identified as a reasonable aim to improve crop yield.
Being the basic organ of photosynthesis, the leaf is constructed in such a way that its orientation and anatomy have a major role in controlling the absorption of light for photosynthesis (Figure 1.3 A). Thus, anatomically, the leaf is highly specialized for light absorption (Terashima and Hikosaka, 1995). For example the epidermis is typically transparent to visible light with convex epidermal cells that act as lenses to focus and intensify the ambient light that reaches the chloroplasts in the lower mesophyll cells. The mesophyll cells themselves are organized to optimize both the capture of light and to enable the influx and efflux of the substrates and products of photosynthesis.
Chloroplasts contain grana are which made up of layers thylakoid membranes (Fig. 1.3 B) that are the sites of light capture by a group of light-harvesting complexes associated with photosystem (PS) II and I. The excitation of special chlorophyll molecules in PSII initiates a series of electron transfers through a number of electron acceptors including plastoquinone, cytochrome b6f complex, plastocyanin and finally to the next PSI (Sacksteder et al., 2000).
Figure 1.3: A typical internal structure of a C3 leaf and a summary of light dependent and independent reactions. (A) shows a typical diffusion path for CO2 and the association between the light reactions, carbon reduction, and photorespiratory cycles (C, Chloroplast; M, mitochondrion; P, peroxisome). (B) depicts the two important processes that take place in the chloroplast. Light-dependent reactions are the energy-capturing reactions in which chlorophyll within thylakoid membranes absorbs solar energy and energizes electrons. The energized electrons move down the electron transport system and are used for ATP and NADPH production. Both ATP and NADPH are used in the light-independent reactions (or simply Calvin Cycle) which take place in the stroma. These are synthesis reactions that use the products of light dependent reactions to fix CO2 into carbohydrate (sugar). Figure (A) is adapted from von Caemmerer and Evans (2010) and (B) from Figure 10.5 of Campbell, Neil A., & Jane B. Reece. Biology. 7th ed. San Francisco: Pearson/Benjamin Cummings, 2005, p. 185).
PSI reduces NADP+ to NADPH through the action of ferredoxin and the flavoprotein ferredoxin-NADP reductase. PSII contributes to the electrochemical proton gradient that results in the generation of ATPs by the action of ATP synthase enzyme. Both products of these light-dependent reactions namely ATP and NADPH will be used in the Calvin-Benson (CB) cycle to reduce CO2.
The CB cycle is a light-independent process located in the stroma of the chloroplast and in general comprises three stages namely, carboxylation, reduction and regeneration (Bassham et al., 1950). CO2 diffuses through the stomata and makes its journey all the way to the stroma of chloroplast in order to react with ribulose 1,5-bisphosphate (RuBP) which is catalyzed by Rubisco to generate the 3-carbon intermediate 3-phosphoglycerate. This intermediate will be reduced to 3-carbon triose phosphates by enzymatic reactions driven by the photochemically generated ATP and NADPH from the light-dependent reactions. Finally the cycle regenerates RuBP molecules to start the carboxylation process all over again. Since Rubisco can have both carboxylase and oxygenase activities (Farquhar et al., 1980), it is crucial to maintain a high CO2 concentration around it to promote carboxylation and this is generally achieved through stomata aperture control (Casson and Hetherington, 2010) and mesophyll conductance (Evans et al., 2009). Otherwise the oxygenase reaction of Rubisco will initiate the undesirable (in terms of crop production) photorespiratory pathway involving two other organelles namely peroxisomes and mitochondria (Fig. 1.3 A).
Photosynthesis occurs at the cellular level and involves a number of biochemical processes. However, despite developing an increasingly detailed picture of photosynthetic mechanisms, research has so far failed to deliver any real gains via improving the basic machinery that have given increased crop yields (Fereres and Connor, 2004). One of the most obvious targets to enhance photosynthesis is by engineering Rubisco, the key photosynthetic enzyme responsible for CO2 fixation. The photosynthetic rate of many C3 crops (such as rice) is limited by the activity of Rubisco which also has an affinity to oxygen that initiates the apparently wasteful photorespiration process (Sharkey, 1988). Various attempts to improve photosynthetic efficiency through Rubisco optimization have shown little progress so far (Andrews and Whitney, 2003; Whitney et al., 2011).
Undoubtedly photosynthesis is influenced by a range of environmental factors such as light, ambient CO2 concentrations and temperature, as well as indirect influences (mediated through stomatal control) to other environmental factors such as humidity and soil moisture (Taiz and Zeiger, 2010a). The dependence of photosynthesis on environmental factors is very significant to farmers and agronomists because crop yield depends strongly on prevailing photosynthetic rates in an ever changing environment. On the other hand, the efficiency of photosynthesis in response to the environment is strongly influenced by leaf physical components such as stomata and the constituent cellular architecture, which are themselves responsive to these same environmental factors.
1.2.1 The role of stomata and improvements in photosynthesis
The major focus of this study is stomata, microscopic pores on the aerial part of terrestrial plant surfaces. The movement of stomata (opening and closing) regulates the exchange of external CO2 for internal O2 and water evaporation (transpiration). Fundamentally, a stoma comprises two guard cells, a pore and underlying airspace (sub-stomatal chamber). The morphology of guard cells are used to classify stomata into two broad classes (Evert, 2006) namely dumbbell-shaped stomata (typical of grasses such as rice, Fig. 1.4 A) and kidney-shaped stomata (in other C3 species, Fig. 1.4 B). Stomata have a very important role in leaf physiology in balancing the need for photosynthetic CO2 uptake against the need to control water loss through transpiration. Evaporation of water from stomata promotes leaf cooling thus transpiration is thought to be important in maintaining leaf temperature within an optimal range (Mahan and Upchurch, 1988). This is important for photosynthesis to take place efficiently because this greatly affects the enzymatically catalysed reactions, as well as membrane processes in photosynthesis (Lambers et al., 2008). Particularly in C3 plant species such as rice, high temperature has a significant effect on the kinetic properties of Rubisco whereby the affinity for CO2 decreases more rapidly than that for O2 (Atkin et al., 2006). Furthermore, at high temperature the solubility of CO2 declines more strongly than does that of O2 (Warren 2007).
Figure 1.4: Stomatal shape comparison between dumbbell-shaped rice stoma (A- typical of grass) and kidney-shaped stomata in Aglaonema sp. (B- typical in other C3 plant species). Scale bars equal 30µm. (Yaapar, unpublished).
The combined elevated temperature effects on CO2 solubility and Rubisco affinity lead to the increase in photorespiration, a process that reduces the efficiency of photosynthesis in C3 plants such as rice. Even so, photorespiration at the same time protects the photosynthetic apparatus under stress conditions. Reactive oxygen species (ROS) molecules are generated to regulate acclimation response to stress (Apel & Hirt 2004). Photorespiration helps minimise ROS production by directly or indirectly using ATP and NADPH. CO2 limitation in the CB cycle (to use excess ATP) and lack of NADP+ or other acceptors in photosystem I (PSI) will lead to generation and accumulation of ROS that must be discarded (Spinola et al., 2008).
In addition, net photosynthesis has been conventionally analysed in terms of stomatal and non-stomatal limitations (Jones, 1985; Ni and Pallardy, 1992). Stomatal limitation results from the resistance to CO2 diffusion into intercellular spaces while the non-stomatal limitation is often assumed as a metabolic constraint. Farquhar and Sharkey (1982) proposed an analysis of the response between the net CO2 assimilation rate and internal CO2 concentration (Ci) in order to study these two limitations separately. In this concept, stomatal conductance (gs) is a useful parameter in studying C3 plant photosynthesis. gs (the inverse of stomatal resistance) is a measurement of the flux of H2O and CO2 through the stomata, in and out of the leaf (Farquhar and Sharkey, 1982). Since CO2 and water share the same stomatal entry pathway, this presents the plant with a functional dilemma, optimizing CO2 uptake while minimizing substantial water loss. After diffusing through the boundary layer on the leaf surface, the stomatal pore is the main port of entry for CO2 into the leaf’s interior. At this point, stomatal conductance/resistance will determine the amount of CO2 that diffuses into the leaf. The relationship between stomatal size and density can be expressed in terms of gs. and can be mathematically obtained (for maximum possible gs) from a model by Franks and Farquhar (2001):
Equation 1.1
Where gs is stomatal conductance to water vapour (minus boundary layer), Sdensity = stomatal density (m-2), D = diffusivity of water in air (m2s-1), Spore= stomatal pore area, V= molar air volume (m3 mol-1), Sdepth= stomatal pore depth (m) and π = 3.142. From this, Franks and Beerling (2009) further proved using fossil samples that high densities of small stomata were the only way to attain the highest gsmax values required in order to counter two major drops in ancient atmospheric CO2 concentrations (from about 3000ppm to 1000ppm) which occurred during Permo-Carboniferous (300-350 million years ago) and Cenozoic glaciations (100 million years ago). This is possible because stomatal diffusivity is inversely proportional to the distance a gas has to travel through the Spore. This value increases with guard cells pair length as they inflate forming an approximately circular aperture.
Interestingly, it has been shown in rice that stomatal conductance is strongly correlated with leaf photosynthesis (Hirasawa et al., 1988). For instance, the indica rice variety Takanari is known for its higher grain yield and dry matter accumulation (Xu et al., 1997). Taylaran et al. (2011) reported that the high-yielding capacity possessed by Takanari was linked to a higher stomatal conductance that was responsible for a high leaf photosynthetic performance. However, it is still unclear whether stomatal conductance alone is the dominant factor that limits photosynthetic rate since other forms of conductance such as boundary layer, intercellular air space and mesophyll are also present (Kusumi et al., 2012).
It is worth noting that getting CO2 into the leaf via improved conductances (stomata, mesophyll etc.) only partially influences photosynthesis since other factors such as leaf nitrogen and Rubisco enzyme content are more dominating (Makino, 2003; Makino 2011) thus gs can be thought of as a co-dominating factor that correlates to photosynthesis. In fact Rubisco specific activity is significantly different between rice and wheat. In wheat, Rubisco Kcat is greater than rice (about 50% higher Vmax for carboxylation) but rice has lower Km (about 20% lower Km for CO2) (Makino et al., 1988). Moreover the specific activity of Rubisco in rice is significantly lower than many other higher plants as well (Table 1 in Makino, 2003). However Rubisco kinetic properties cannot be a target for classic breeding among rice cultivars because no variability has been found among old and modern rice cultivars (Makino et al., 1987).
1.3 Understanding the rice leaf
1.3.1 General Morphology
In rice, the phytomer concept is used to describe repeating units of vegetative growth. A phytomer comprises a leaf subtended by an internode. A tiller bud is present at the lower end of the internode and a root band is present at both the upper and lower end of the internode (Fig. 1.5). A rice node includes the nodal plate as well as the base of the internode above it that bears the next higher leaf.
Figure 1.5: A rice phytomer units comprises one internode, a node, emerged leaf, tiller bud and root primordia. (Based on a drawing by Hoshikawa, 1989).
On a rice stem or culm, rice leaves are borne alternately on opposite sides. Each leaf consists of two parts, a lower leaf sheath and an upper lamina (leaf blade) (Fig. 1.6 A). These regions are connected by a joint known as the collar. The collar bears a pair of auricles (ears) and a ligule (tongue), protrusions from the leaf surface (Fig. 1.6 B). These structures at the joint are commonly used to distinguish between rice varieties and other grasses (Moldenhauer and Gibbons, 2003). For example, the ligule of O. sativa is long and soft but it is short and tough in O. glaberrima (OECD, 2006).
The leaf sheath is attached to the nodal plate. It wraps around the culm and has a slight swelling just above the node referred to as the sheath pulvinus (Fig. 1.6 C).The sheath is photosynthetically active and encloses both the developing new leaves and the panicle (the terminal shoot of a rice plant that produces grain). During vegetative growth the sheath provides support to the plant and acts as a storage site for starch and sugar (Moldenhauer and Gibbons, 2003). During the reproductive stage of development it mechanically supports the stem by contributing 30-60% toward shoot breaking strength (Chang, 1964).
Figure 1.6: Rice leaf and its component structures. (A) Culm and leaf sheath; (B) Collar, a pair of auricles and ligule; (C) Sheath pulvinus.
The lamina in general is flat and lanceolate in shape and provides the main area for light absorption and photosynthesis. Many parallel veins run along the upper surface of the blade with a prominent midrib on the underside of the leaf. Different rice varieties vary in terms of blade length, width, thickness, area, color, angle and pubescence (the presence of cover with fine, soft and short hairs). Some of these characters have been selected by the breeders to ease work in rice fields. Genetically, there is variation for leaf length and width among cultivars (Jennings et al., 1979). The length of the entire leaf (sheath and lamina) also increases with successively higher position on the main culm (Hoshikawa, 1989).
1.3.2 Leaf anatomy
The rice leaf has a classic arrangement of a monocot leaf in which a regular array of parallel veins occurs. A transverse section of a rice leaf shows that it has both small (SV) and large vascular bundles (LV), with the latter occurring approximately once for every six smaller bundles. Each vein is enclosed in a well-defined bundle sheath (BS) that is linked to both the upper and lower epidermis by fibrous bundle cells (Evans and Caemmerer, 2000).
The rice leaf possesses a specialized type of epidermal cell called bulliform or motor cells (BC). Located between the vascular bundles on the adaxial epidermis, they function to control the rolling of the lamina (Fig. 1.7). Under conditions of water deficit they lose turgor and constrict, causing the lamina to fold or roll inward (Dickison, 2000). They have large vacuoles and little or no chlorophyll (Mishra, 2009). Stomatal files in rice are located next to the veins on the leaf surface. In addition, the rice leaf has two kinds of trichomes, namely micro and macro hairs. Micro hairs are located along the stomatal files or besides bulliform cells while macro hairs are located on silica cells over a thin vascular bundle (Kobayashi et al., 1997).
The rice leaf is generally thinner (about 75µm for IR64 when grown under high-light condition, Narawatthana, 2013) compared to Arabidopsis (about 210µm for Col-0 when grown under high-light conditions, Wuyts et al., 2012). There is no mesophyll cell differentiation into palisade and spongy parenchyma, thus there is no remarkable distinction between the adaxial and abaxial sides (Lafitte and Bennett, 2002). In dicots the internal mesophyll cells are polarized in which the closely packed palisade cells are below the adaxial epidermis and loosely packed spongy cells border the abaxial epidermis (McConnell and Barton, 1998). In rice and some grasses like maize, there is a uniform configuration without polarization, which is termed isobilateral mesophyll (Fahn, 1990).
Figure 1.7: Transverse section of a rice leaf. (LV) large vascular bundle; (SV) small vascular bundle; (BS) bundle sheath cell; (BC) bulliform cell; (TRC) trichome; (STO); substomatal chamber.
1.3.3 Leaf development in rice
The staging of rice leaf development is mainly described by Itoh et al. (2005) and this is summarized below. In defining leaf development it is common to use the plastochron as a unit of developmental time. A plastochron is defined as the interval between two successive leaf initiation events at the shoot apical meristem (SAM). This allows leaves to be allocated a plastochron number (Pi) which defines a specific developmental stage. Table 1 summarizes the staging of adult leaf development in rice.
Table 1.1:
Staging of adult leaf development in rice. I1, Incipient primordium; P1-P6, Plastochron 1-6; (Table is adapted from:
Stage | Name | Events and Descriptions | Figures | |||||||
I1 | Formation of leaf founder cells | Recruitment of leaf founder cells to become leaf primordium but not distinguishable morphologically from other cells in the SAM. Can be detected with the expression of gene OsPNH1 (OryzasativaHOMEOBOX1) (Nishimura et al. 2002). Leaf founder cells are distributed in a half-ring fashion around the SAM. | Blue region (left) and arrowhead (right) indicate a group of founder cells. | |||||||
P1 | Formation of leaf primordium | Protrusion of primordium on the flank of the SAM or crescent-shaped primordium.Elongation of leaf margin around SAM. | Crescent-shaped primordium (left) and P1 is marked with a blue. | |||||||
P2 | Hood-shaped primordium | Hood-like shape. Overlapping of two margins.Differentiation of vascular bundle. | P2 is marked with blue (left). SA, shoot apical meristem. | |||||||
P3 | Formation of ligule primordium | Formation of ligule primordia and (mainly) leaf blade/sheath boundary. The leaf margins overlap and completely enclose the SAM.Differentiation of sclerenchymatous cells.Initiation of epidermal specific cells and small vascular bundle formation occur basipetally. | Arrow indicates blade sheath boundary | |||||||
P4 | Rapid elongation of leaf blade | Differentiation of epidermal speCific cells (bulliform cells, silica cells, cork cells and stomata).Elongation of leaf blade due to the high activity of intercalary meristem at the base of lamina (Kaufman, 1959). | ||||||||
P5 | Rapid elongation of leaf sheath | Elongation of leaf sheath.Emergence of leaf blade from the sheath of proceeding leaf.Formation of lacunae and the maturation of leaf epidermal cells | Cross section around shoot apex; asterisk, lacuna. | |||||||
P6 | Maturation | Bending of leaf blade at the lamina joint (collar) signifies leaf maturity and complete growth. | Mature leaf (left); Cross-section of a leaf (right); LB, leaf blade; LG, ligule; AU, auricle; LJ, lamina joint; LS, leaf sheath; LV, large vascular bundle; SV, small vascular bundle; SC,sclerenchymatous cell; phloem; XY, xylem; BS, bundle sheath cell; BC, bulliform cell; |
1.3.4 Stomatal development
As mentioned in Fig. 1.4, stomata occur in two classes namely dumbbell (as in rice) and kidney-shaped (as in Arabidopsis). Each form follows a unique developmental series and distribution pattern. Despite this, in general both systems involve asymmetric and symmetric cell divisions of specialized epidermal cells. In both types the process is initiated by the asymmetric division of a protodermal cell (Fig. 1.8B), that gives rise to a meristemoid, which is a transient cell state of the stomatal lineage. A meristemoid then proceeds to differentiate into a guard mother cell (GMC) that eventually undergoes a single symmetric division to produce a pair of guard cells (GCs). This is only a simplified explanation for both stomatal developmental systems, with each having its own elaborate characteristics for every transitional state until maturity (reviewed by Bergmann and Sack, 2007 and described below). Even though both systems share a basic common ground in stomatal development, the leaf in Arabidopsis is not clearly zoned. The more mature stomata are located within the tip region but this is not absolute since sister cells of a stomata precursor can undergo division later in time. This intercalates newly formed stomata among stomata that have been previously formed. In rice, the leaf is strongly zoned so that the base is the youngest and in this cell proliferation zone the epidermal files that can later form stomata are established (Liu et al., 2009).
Much of our understanding about stomatal development comes from the studies using the model dicot plant Arabidopsis thaliana. In this plant, the stomatal lineage begins with a subset of protodermal cells becoming a meristemoid mother cell (MMC – Fig. 1.8B- light blue). The MMC undergoes an asymmetric division, producing a small meristemoid (Fig. 1.8B- yellow oval) within a large sister cell (irregular white shapes) called a stomatal-lineage sister cell. The meristemoid undergoes a cell-state transition to produce a GMC and it divides once symmetrically to produce a pair of GCs. Specialized GCs work in concert to control stomatal opening and they cease division. Liu et al. (2009) also proposed the sites of action of three important regulators that direct sequential stomatal development steps seen in Arabidopsis (Fig. 1.9A).
Figure 1.8: Key differences in morphological and developmental pattern between Arabidopsis thaliana (dicot) and Oryza sativa or rice (monocot) stomata. (A) Kidney-shaped stoma of Arabidopsis and dumbbell-shaped stoma of rice. (B) Stomata distribution and developmental pattern in Arabidopsis (top) and rice. GMC (orange oval in Arabidopsis, yellow block in rice); SC (small dark blue block in rice); GC (red kidney in Arabidopsis, red dumbbell in rice). Figure adapted from Liu et al. (2009).
Figure 1.9: Schematics of comparison between stomatal development in Arabidopsis (A) and rice (B) with proposed sites of expression of three closely related bHLH transcription factors SPCH, MUTE and FAMA. In rice, it is proposed that the action of SPCH would occur before the stomatal cell row stage. Figure adapted from Liu et al. (2009).
These three positive regulators are the closely related basic helix-loop-helix (bHLH) domain transcription factors namely SPCH (SPEECHLESS), MUTE and FAMA (MacAlister et al., 2007). SPCH controls the initial asymmetric division of protodermal cells, giving rise to the stomatal lineage namely meristemoid. Later, MUTE is required to set GMC fate from the meristemoid stem cell. Eventually, FAMA controls the symmetric division of the GMC into a pair of guard cells (Ohashi-Ito and Bergmann, 2006).
In rice, stomatal development occurs in a more fixed manner (specific epidermal files) compared to the scattered patterning seen in Arabidopsis. Generally, stomatal development in rice can be divided into five stages (Stebbins, 1960) and the stomata in rice start as a row of cells, known as a stomatal cell row (Fig. 1.8B- light blue shading). These rows can be adjacent but are normally separated by non-stomatal epidermal cell rows. This stomatal cell row undergoes cell division to produce stomatal precursors, which undergo asymmetric division to produce GMCs (Fig. 1.8B- yellow blocks). GMCs induce the neighboring cells to produce subsidiary cells (SCs) (Fig. 1.8B- small dark blue blocks). Later GMCs divide symmetrically to produce a pair of GCs that undergo extensive elongation and morphogenetic changes as they mature (Fig. 1.8B, small red dumbbells). Each stomatal complex (Fig. 1.10) comprises two GCs that are narrow with thickened walls and two subsidiary cells flanking the guard cells.
Even though morphology and ontogeny between Arabidopsis and rice stomata are reasonably different, the protein sequences of the switches (SPCH, MUTE and FAMA) that control major cell fate transition during stomatal development are highly conserved between these two angiosperms groups. However, their functions, especially for MUTE, SPCH1 and SPCH2, are somewhat divergent (Liu et al., 2009). In Arabidopsis, the functions of SPCH and MUTE are largely tied to the initiation and termination of the stem cell-like division in meristemoids but in grasses it is hypothesized that the activity of OsMUTE could be a hybrid between that of Arabidopsis SPCH and MUTE (Fig 1.9B). There is evidence that OsMUTE is expressed during early stages in which stomatal files are forming (MUTE in Arabidopsis is expressed in later stages where meristemoids have formed). On the other hand, FAMA function is conserved between dicots and monocots despite their different stomatal morphologies.
Figure 1.10:
SEM micrograph of a mature stomatal complex cross section in rice and its associated microstructures such as hautegelenke or hinge and substomatal cavity (Yaapar, unpublished)
Despite the progress made in elucidating the molecular control of stomatal differentiation, there are still many blanks to be filled in, especially involving rice, the C3 model as well as Setaria sp. the C4 model for monocot plants. For example, it is still unknown when SPCH is expressed in rice, although it has been proposed by Liu et al. (2009) that the action of SPCH would occur before the stomatal cell row stage (Fig. 1.9). Furthermore, research in Arabidopsis has confirmed the presence of integrators SCREAM (SCRM) and SCRM2, two bHLH proteins that work along with SPCH, MUTE and FAMA by promoting all three cell-state transitions (Hunt and Gray, 2009). The current model suggests that the heterodimerization of SCRM/SPCH enhances activation of stomatal lineage entry but the specific mechanism underlying heterodimer stabilization still remains unclear, even in Arabidopsis (Kanaoka et al, 2008). This shows that much more extensive work is required to also elucidate the stomatal three-cell state transitions in grasses.
1.4 Environmental signals affect leaf and stomatal development
Many of the key developmental events of a plant’s life cycle such as germination and leaf formation, are regulated by environmental signals such as temperature and light. Undoubtedly genetic control plays the most important role in determining leaf size, structure and shape but these parameters are flexible to a certain degree. This is because leaves are able to tailor their growth based on environmental conditions (Tsukaya, 2006).
1.4.1 Light as a stimulus
Light is one of the most important environmental factors that regulates the development of the photosynthetic apparatus in vascular plants. In a high light or low light environment, plants develop sun or shade leaves, respectively (Boardman, 1977). The extent to which a particular species can alter it’s phenotype in response to growth in different environments, or respond to a change in environment is known as phenotypic plasticity and the processes by which this occurs is known as acclimation. Generally, high-light responses occur to maximize light-saturated rate of photosynthesis whilst low-light responses occur to optimize photon capture. This is reflected in the general fact that sun leaves are thicker, smaller, with more developed palisade tissue and higher stomatal density on both adaxial and abaxial surfaces when compared with shade leaves (Anderson and Osmond, 1987; Murchie and Horton, 1997). (A more detailed discussion of the characteristics of sun and shade leaves is provided in Chapter 3.)
In addition, irradiance levels also affect stomatal density. It has been shown in Arabidopsis that a high light environment received by the older leaves will result in greater stomatal density on both adaxial and abaxial sides of a newly formed leaf (Coupe et al., 2006). This phenomenon shows that stomatal density is one of the contributing factors that explains why high-light grown leaves possess higher photosynthetic capacity. In a recent experiment, Tanaka et al. (2013) demonstrated that increased stomatal density in STOMAGEN-overexpressing Arabidopsis, enhanced the photosynthetic rate by 30%. The STOMAGEN gene encodes a small peptide with a putative secretory signal sequence at its N-terminus and is expressed preferentially in mesophyll cells and is a positive regulator of stomatal development in Arabidopsis. Even though higher photosynthesis generally results from higher gs, this is not translated into efficient water use because STOMAGEN overexpression did not increase whole plant biomass, although it’s silencing did cause a reduction in biomass. In their work, Tanaka et al. (2013) showed that improved photosynthetic capacity, through higher stomatal density, can be achieved by modulating the gas diffusion process in the leaf. This is regarded as a potential trait for plant engineering in order to improve photosynthesis.
Despite these promising findings, the relationship between stomatal characteristics, photosynthesis and yield for rice has yet to be established (Kundu and Tigerstedt, 1999; Miskin et al., 1972). Low stomatal density in rice has been shown to be attainable only when low irradiance level is coupled with elevated CO2 but low-irradiance conditions alone, is adequate to produce small stomata (Hubbart et al., 2003). Moreover, the effect is more prominent on the adaxial surface thus validating a previous publication (Zhang et al. 2009) and such specific abaxial-adaxial-stomatal response has been shown in Arabidopsis (Lake et al., 2002) and common bean (Wentworth et al., 2006) as well. Since high and low light-grown rice leaves will produce sun and shade leaves respectively, it is hypothesized that stomatal distribution and size should vary accordingly to photosynthetic capacity. These stomatal characters are believed to be set during development by the irradiance level received by older leaves, although the exact developmental stages at which these characters are determined is still unknown. Nevertheless it is worthwhile mentioning that developmental and dynamic acclimation are distinct processes (Athanasiou et al., 2009). Dynamic acclimation is especially important in determining the fitness of plants growing in changing environments, for example, their ability to change the photosynthetic capacity of developed leaves. Developmental acclimation involves changes in leaf morphology and composition (for example stomata which are the focus in this study) being optimized for the conditions (for example low or high light) experienced by plants. Thus measurements made reflect conditions experienced as the leaves develop.
1.4.2 Systemic signalling and stomatal development
Ferjani et al. (2008) summarized in their review, that leaf differentiation into sun or shade types is regulated remotely by mature leaves via long-distance signaling. They also highlighted that light quantity is the major stimulus, triggering a systemic signal that controls leaf development. They suggest that the known photoreceptors such as phytochromes, cytochromes and phototropins are probably not involved in the signal perception leading to sun and shades leaves. The exact light sensory mechanism is still unknown but several candidate signals have been considered (Karpinski, 1999; Koch, 2000, 2004). The work by Murchie et al. (2005), using the fifth leaf in rice as a model system, showed that rice grown in a low irradiance (200 µmol m-2 s-1) environment and a high irradiance (1000 µmol m-2 s-1) environment would produce sun/shade leaves respectively whereby sun-type rice leaves had thicker leaves, higher light-saturated rates of photosynthesis (Pmax), higher amounts of Rubisco protein, and a lower chlorophyll a/b ratio. However, photosynthetic acclimation is limited by leaf age. For instance transfer from low to high light during full leaf extension only altered chlorophyll a/b but not Rubisco protein content.
The development of stomata relies on many exogenous and endogenous signals including light, CO2, temperature, water availability, abscisic acid content and the activities of cellular regulatory proteins and RNAs (Bergmann and Sack, 2007; Kwak et al., 2008;). However, much of the work done has used Arabidopsis as the model plant, thus detailed information in rice or grass for that matter is still lacking. For example, a stomatal density study by Coupe et al. (2006) demonstrated that when mature Arabidopsis leaves were given high or low light treatment, the young, developing leaves that were not receiving the treatment grew with stomatal density as if they were exposed to the treatment. They found that mature leaves that received high light treatment resulted in a newly formed leaf with higher stomatal density on both adaxial and abaxial surfaces. Arabidopsis leaf stomatal density is lower on the adaxial surface but in rice the stomatal density is believed to be about the same on both sides of the leaf since the leaf grows in an erect position. Moreover it has been shown in rice that the alteration of stomatal properties (size and density) is one of the strategies in acquiring photoacclimation through systematic signalling generated in mature leaves (Hubbart et al., 2013). Interestingly, both irradiance levels and CO2 concentrations affect stomata differently in rice where low-light condition produces smaller stomata than high-light condition but when coupled with higher CO2 environment this effect is attenuated. Similarly for stomatal density where under low-light condition it is lower than high condition only when higher CO2 environment is introduced. This suggests that there is a complex interactions at play to confer the most beneficial stomatal morphology in rice acclimation.
1.5 Research project outline
1.5.1 Aims
1. Characterise the pattern of stomatal differentiation in rice leaves grown under different levels of irradiance.
2. Investigate the outcome of irradiance-induced altered stomatal patterning on rice leaf photosynthetic performance.
3. Identify the stages of rice leaf development during which altered irradiance can influence stomatal patterning.
4. Investigate the outcome of developmental-stage specific altered stomatal patterning on rice leaf photosynthetic performance
5. Develop a whole mount in situ hybridisation method for the analysis of gene expression in rice leaves with the ultimate aim of characterising stomatal gene expression during early rice leaf development.
1.5.2 Overarching hypotheses
1. There is a specific phase in development when a rice leaf can respond to altered irradiance by altering stomatal patterning, leading to altered stomatal density and size in the mature leaf.
2. Altered stomatal properties in in the mature rice leaf alters leaf photosynthetic performance.
3. Whole-mount in situ hybridisation can be used as a rapid and reliable method to study stomatal gene expression.
Chapter 3 Aims
1. Characterise the pattern of stomatal differentiation in rice leaves grown under different levels of irradiance.
2. Investigate the outcome of irradiance-induced altered stomatal patterning on rice leaf photosynthetic performance.
Chapter 4 Aims
1. Identify the stages of rice leaf development during which altered irradiance can influence stomatal patterning.
2. Investigate the outcome of developmental-stage specific altered stomatal patterning on rice leaf photosynthetic performance
Chapter 5 Aims
1. Develop a whole mount in situ hybridisation method for the analysis of gene expression in rice leaves with the ultimate aim of characterising stomatal gene expression during early rice leaf development.
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