Introduction

Increasing understanding of the many molecular and biochemical processes that respond in a purposive way to the changing environment has given rise to an appreciation that many, if not all, environmental cues evoke primary responses at a molecular level, and that it is these responses that result in changes in gross plant physiology and morphology. Likewise, changes in the relative proportions of metabolites and ions within intracellular compartments in response to such environmental cues also give rise to multiple changes in gene expression. The interaction between these levels of complexity in response to changes in the external environment is illustrated in Scheme 13.1.

This chapter describes and discusses approaches: (1) for the unbiased analyses of gene, protein and metabolite function facilitated by a variety of high-throughput approaches; and (2) for the focused analyses of specific genes, gene products and metabolites. The former approaches seek to identify hitherto unknown genes and molecular interactions, while the latter are used to probe those elements that we currently consider most important in understanding and interpreting how photosynthetic processes relate to ecophysiological questions. In particular, we discuss aspects of the isolation and assay of the carboxylating enzymes, Rubisco and phosphorenolpyruvate carboxylase (PEPC), owing to their pivotal roles in assimilation and to the continuing interest in their measurement. In general, we have selected methods and approaches that have been applied in our laboratories, but acknowledge that many alternative methods could have been described, which are equally reliable and quantitative.

The Development of Gas-Exchange Systems and their Practical Applications

Gas-exchange systems have come a long way since the technology for real-time measures of gas concentrations in air became available. The method of choice among most plant biologists involves the use of IR gas analysers (IRGAs) integrated into gas-exchange systems to measure concentrations of CO2 taken up by photosynthesis and water released via transpiration (E) over a range of conditions that can be manipulated by the researcher. Typically, gas-exchange systems rely on steady state conditions surrounding photosynthetic tissues, however, unique systems have been developed to measure non-steady state gas exchange for rapid responses (e.g., seconds and faster) to changing conditions (e.g., Laisk and Oja, 1998). Less common, but never-the-less available and quite useful, are methods that rely on oxygen analysers to measure photosynthetic oxygen release. Furthermore, there are methods for determining rates of photosynthesis that rely on Chl-F, reflective indices (e.g., photochemical reflectance index, PRI) and isotopic analysis, each of which are discussed in other chapters. The objective of this chapter is to present and discuss the current methods specific to gas exchange for measuring photosynthesis. This objective includes discussions on the current state of technology, a summary of the equations and theories behind the measurements, potential sources of error and the variables of interest that can be extracted from the most common measurements. Although the focus of this chapter is to provide an ‘entry point’ into the common gas-exchange techniques, it is not intended to provide a complete description of all gas-exchange systems and all physiologically meaningful data that can be collected. Rather, the reader is recommended towards a wide variety of excellent texts that detail many aspects of gas exchange (e.g., Long and Hällgren, 1993; Laisk and Oja, 1998; Long and Bernacchi, 2003).

Introduction

The appearance of the C4 photosynthetic pathway in the Earth’s flora represents one of the most impressive and curious examples of evolutionary diversification and biogeographic expansion in the history of life (Ehleringer and Monson, 1983). This complex pathway, involving novel patterns of biochemical compartmentation and anatomical design, has evolved with independent but convergent patterns approximately fifty times during the relatively short geological span of 12–15 million years (Kellogg, 1999; Monson, 1999; Sage, 2004; Christin et al., 2007). The appearance of C4 photosynthesis has changed the nature of photosynthetic productivity and ecosystem structure on Earth, both regionally and globally. Grassland ecosystems emerged in southwestern Asia, Africa and North America during the mid- to late-Miocene (5–10 Ma) and continued through the Pliocene, (~3Ma), with many of these systems dominated by C4 species (Cerling, 1999; Beerling and Osborne, 2006). During the appearance of C4 grasslands, the trophic structures of grazed ecosystems were completely revised, resulting in the emergence of novel mammalian lineages (Cerling et al., 1993; Wang et al., 1994; MacFadden and Cerling, 1996; Ehleringer et al., 1997). Arguably, there is not a better example in the history of life to illustrate the tightly integrated nature of evolutionary novelty and ecological impact, as that shown in C4 photosynthesis. Clearly, C4 photosynthesis, though present in only 8,000 of the estimated 250,000 higher plant species, deserves a significant role in the discussion of plant biology.

Introduction

The evolutionary traits described in the previous chapter are common to all photosynthetic types, and evolved originally in C3-like species. Under current atmospheric conditions, the O2 inhibition of photosynthesis occurs through oxygenation of RuBP and subsequent loss of CO2 through the reactions of photorespiration in C3 plants. Consequently, a very significant part of photosynthetic evolution in vascular plants has been the development of mechanisms for reducing photorespiration by concentrating CO2 around Rubisco, thus returning this enzyme to an atmospheric condition that resembles the primitive earth. These CO2-concentrating mechanisms are known as CAM and C4 photosynthesis, while all other plants are referred to as C3. Other CO2-concentrating mechanisms exist in some algae and cyanobacteria (Kaplan and Reinhold, 1999; Raven et al., 2008), but these will not be discussed here.

The evolutionary origins of CAM and C4 are presumably tied to changes in paleoclimates and atmospheres, particularly to historic variations in CO2 and O2, and locally warm climates (Fig. 24.2 in previous chapter). Under higher CO2 partial pressure and/or lower temperature the C3 pathway fixation does not exhibit limitations that would put a premium on coupling it with a CAM or C4 pathway. The origins of vascular plants date to around the mid-Silurian (~440 MA, Fig. 24.1 in previous chapter) and these plants were likely C3, although the astomatous CAM plant Stylites andicola has been suggested as a possible model of early plant evolution (Keeley et al., 1984). Coupling the C3 pathway with one of the CO2-concentrating mechanisms, CAM or C4, occurred subsequent to the emergence of vascular plants and possibly arose more than once over the past 400 million years.

Introduction: Why Use Photosynthesis Models?

One of the goals of science is prediction, and quantitative predictions are based on models. Models vary in the degree to which they represent underlying processes from purely empirical regression models to those that represent physical, chemical and biological processes (mechanistic or process models). Mechanistic models tend to be more complex and are empirical at the level of the processes they include (parameters). Mechanistic models can also test whether our current knowledge is sufficient to explain experimental data and generate and test new hypotheses that lead to progress in understanding. Prediction of carbon fixation of vegetation by photosynthesis in a changing environment is important to assess ecological matters like global climate change or agronomic issues such as yield improvements. Models of photosynthesis play a key role in predicting primary production of vegetation and crop yield in a variable climate. They have also been implemented in weather-forecast models, improving predictions of humidity and temperature anywhere from 5 to 10% (Goudarzi, 2006). The level of detail of these models is largely dependent upon the objectives of the studies for which the models were designed. Questions concerning effects of environmental conditions on the process of photosynthesis itself can be addressed using single-leaf photosynthesis models. Vegetation canopies are composed of leaves and of support structures (stems, trunks) that represent a greater challenge for predicting photosynthesis. The simplest canopy models invoke the so-called ‘big-leaf’ assumption, and may include various simple approximations for structural and physiological complexity (e.g., Sellers et al., 1992). More complicated models include multiple canopy layers each with different properties (e.g., Medlyn et al., 2005). Layered models require detailed knowledge of the variation in parameters throughout the canopy depending on position and age, as well as knowledge of the distribution of foliage and environmental variables in the canopy. The objective of modelling is double. On one hand, models can be used in a bottom-up approach to predict photosynthesis variations in response to environmental variables. To do so, it is needed to first parameterise the photosynthesis model itself, obtaining representative values of the maximum carboxylation efficiency (Vc,max), the maximum rate of electron transport (Jmax) and the capacity for TPU (sections 8.3–8.6).

Introduction

Photosynthesis is typically understood as the light-dependent production of sugar from carbon dioxide (CO2). The endosymbiotic chloroplast is the cellular location for most of this metabolism in plants, but some additional metabolism occurs in the cytosol to make the sugars that will be transported around the plant, mainly sucrose and also sugar alcohols, such as sorbitol and manitol. There are many processes that can properly be called photosynthesis, but a core set of processes underlie most of the considerations in this book. This chapter will provide an overview of those processes, and many topics covered in this chapter are the subject of more in-depth chapters later on. This chapter begins by describing the initial capture and temporary storage of light energy as highly reactive molecules (nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine triphosphate (ATP)) on carbon. By reducing (i.e., by adding electrons to) carbon from its most oxidised state (CO2) to the status of sugars (CH2O)n, the energy initially stored as NADPH and ATP can be stored on the carbon. Additional energy can be stored on each carbon atom by reducing it fully, as happens in the synthesis of oils (R-CH2-R), but this is generally not considered when describing photosynthesis. Finally, issues surrounding uptake of the CO2 will be addressed.

Photochemistry Synopsis

Photochemistry, the capture of light energy and its conversion to chemical energy suitable for reducing CO2 to sugar, is the source of nearly all energy available to living things. Energy captured by absorbing molecules is stored as the high-energy intermediates NADPH (reducing power) and ATP (sometimes called the energy currency of the cell).

Basic characteristics of solar radiation

Among the factors affecting plants, solar radiation is perhaps the most heterogeneous in space and time. Important parts of solar radiation provide energy for photosynthesis and serve as signals in photoregulation of plant growth and development. The sun radiates energy in the spectral range from 280 to 4000 nm, with a maximum in the blue-green (480 nm; Fig. 16.1). Within the PAR, solar radiation peaks at ca. 590 nm (Fig. 16.1). Solar radiation can be segregated into direct solar radiation and diffuse sky radiation, which reaches the ground after multiple scattering on atmospheric particles and clouds, reflection from the ground surface and additional scattering in the atmosphere (Ross, 1981).

The widespread, albeit vague, term light is used for the portion of the electromagnetic spectrum in the vicinity of visible light (Kohen et al., 1995). Many past ecological and physiological studies were based on measurements that represent the stimulation of the human eye by radiant energy, a measure called illuminance and expressed in foot-candles (English system) or luxes (metric system). The human eye is most sensitive in the green spectral region, centered around 550 nm, whereas any quanta in the spectral region of 400–700 nm have enough energy to drive photosynthesis, so illuminance is obviously not well suited for plant science.

Introduction

Photosynthesis studies and models are usually restricted to green leaves. However, a closer observation of nature reveals that most plants also photosynthesise through non-foliar structures, and photosynthetic tissues are not always green. Non-foliar photosynthesis is performed by a variety of organs such as stems, bark, roots, petioles, fruits or flowers. These structures contribute positively to the carbon balance, and in some leafless species these are the only photosynthetic organs. In general, two main groups of photosynthetically active organs can be discerned (Aschan and Pfanz, 2003): those optimised for photosynthetic performance that achieve a net positive carbon gain (such as green petals, leaves or stems); and those involved in the internal recycling of CO2 released by respiration (such as roots, fruits or chlorophyll-containing bark) (Table 7.1). With a few exceptions, chlorophyll is the only green pigment in plants, implying that all green tissues are chlorophyllous, and all chlorophyll-containing organs are, to some extent, photosynthetic. Nevertheless, photosynthetic organs may not appear green externally because of chlorophyll being masked by outer layers of pigmented cells, waxy cuticles or bark. In addition to plants with whole leaves or parts of leaves (variegated leaves) presenting special photosynthetic characteristics, unique photosynthetic adaptations are also shown by parasitic and submerged plants. Also, stomatal cells from typical leaves of higher plants display specific photosynthetic features, different from those of mesophyll cells.

Introduction

The ‘tropics’ are the most diverse and productive ecosystems on earth. They are broadly defined as those ecosystems that lie between 23.4° N and S latitude. The development of tropical ecosystems as we know them occurred shortly after the rise to dominance of angiosperms and coincided with the diversification of many angiosperm lineages. During this period, between 100 and 36 MA, warm paleoclimates supported much larger areas of tropical vegetation globally. Tropical forests were found not only in equatorial regions but also in mid latitudinal bands north and south of subtropical arid belts (Morley, 2000). Tropical climates are broadly characterised as having more diurnal than seasonal variation in temperatures, but often large seasonal variations in rainfall. Within the tropics, ecosystems range from hot, humid-lowland wet-tropical rain forests to cold, dry high-alpine paramos. Figure 27.1, for example, shows 30-year monthly means for temperature and precipitation for four stations within 1°C of the equator. The Kenyan station had the greatest temperature variations, but even then, the range was only 4°C. Izobamba, Ecuador, at 3050 m elevation, was nearly 15°C cooler than the other sites. Rainfall totals varied eightfold between Garissa, Kenya and Padang, Indonesia, and even within Indonesia precipitation varied by 40% between the sites.

Although tropical plants face similar basic ecophysiological challenges to those in non-tropical ecosystems, the tropics are unique in the sheer diversity of species and life forms and the general lack of freezing temperatures (except in high-mountain ecosystems). In this chapter, we will concentrate our discussion on three major types of tropical environments (wet tropical forests, savannas and mangroves). In each ecosystem, we explore major areas of research related to the ecophysiology of photosynthesis.

INTRODUCTION

General aspects of plant responses to nutrients

Plant growth requires the incorporation of elements (nutrients) into plant organs. In non-woody plants, 15–20% of fresh weight is made from such elements, the rest being water. There are two criteria to consider an element as essential. First, an element is essential if a plant cannot complete its lifecycle (till viable seeds) in its absence. Second, an element is essential if necessary to synthesise molecules that cannot be replaced by other element(s) (for example, N in proteins). In natural ecosystems, soil-nutrient availability is rather heterogeneous, and plants may adapt their growth to nutrients taken up by roots exploring a determined soil volume. In agricultural areas, the situation is different. Lack of or excess nutrients are frequent, owing to soil characteristics (which may immobilise nutrients) or to growers’ applications, respectively.

An idealised representation of plant growth-rate response to availability of any given nutrient would show three different zones: namely (1) deficient; (2) adequate; and (3) toxic. In the range of low nutrient concentrations (deficient zone), growth and the plant nutrient concentration markedly increase as soil nutrient availability increases. As availability increases further the so-called critical concentration is reached. This corresponds to the lowest concentration of nutrient in plant tissue that gives almost maximal growth. Above this point, increases in soil nutrient availability do not affect growth (adequate zone). In the adequate zone, there is a plentiful supply of nutrients, and the excess nutrients may be taken up and stored in leaf vacuoles, special storage proteins in bark or uptake may be downregulated so as to avoid taking up excess nutrients. This zone is fairly wide for macronutrients, but narrower for micronutrients. If nutrient availability increases more and uptake cannot be controlled, toxicity appears and growth is reduced (toxic zone).

The phrase global change is generally associated with alterations of climate (temperatures, fluctuations in precipitation, etc.) that stem from changes in atmospheric composition. In reality, global change also encompasses more than changes in climate or atmospheric composition; any global-scale change that influences biota directly or indirectly can be considered global change. Global change has influenced the biosphere throughout geological time, with changes occurring over periods that allow for either species to evolve to these changes when they occur over long periods, to adapt or acclimate to the changes or to perish when neither of the previous two responses is effective. Although we are currently in the midst of abrupt global change, it certainly is not the first time that rapid global change has occurred. What differentiates the current changes to our planet from all other global-change events is that these abrupt changes are brought about through anthropogenic influences and that they are occurring more rapidly than in previous occasions.

One of the major challenges of a chapter focused on plants and global change is that the topic is extensive. Many chapters of this book focus on photosynthesis responses to most of the predicted global-change scenarios, and entire volumes can be devoted to these and other global-change scenarios. Further, many reviews have been published that address photosynthetic responses to single environmental-change factors (Ceulemans et al., 1999; Saxe et al., 2001; Ainsworth et al., 2002; Long et al., 2004; Ainsworth and Long, 2005; Hikosaka et al., 2006; Sage and Kubien, 2007; Wittig et al., 2007). Our goals in writing this chapter are: (1) to provide a synopsis of global-change trends; (2) to identify the potential impacts of these trends on photosynthesis; (3) to identify the potential for photosynthesis to mitigate global change; and (4) to present ecosystem and general-circulation models as predictors of the interactions between plants and global change.

Introduction

Remote sensing of photosynthesis consists of directly measuring or indirectly estimating photosynthesis rates or photosynthesis-related parameters using non-contact devices positioned far from the plant or canopy. This definition is a broad one, and includes the gas-exchange measurements at the ecosystem level described in Chapter 14, but the term is more commonly used to describe radiation measurements with spectrometric techniques. Three main groups of spectrometric techniques have emerged to remotely assess photosynthesis or photosynthesis-related parameters: reflectance, fluorescence and thermal imagery (Fig. 15.1). Each of these principles has advantages and inconveniences as compared with the others. These are summarised in Table 15.1, and will be discussed in more detail throughout the chapter.

Remote sensing has been identified as an essential tool to complement gas-exchange and atmospheric-circulation models, in order to monitor accurately the spatial and temporal changes in biosphere primary production (Sellers et al., 1992, 1997; Baldocchi, 2008; Malenovsky et al., 2009). Although there have been important advances in this field during the past thirty years, a comprehensive review of the techniques available and their scope is lacking. Partial reviews on remote sensing of reflectance (Peñuelas and Filella, 1998; Gamon and Qiu, 1999), fluorescence (Cerovic et al., 1999 ; Moya and Cerovic, 2004) and thermometry (Jones, 1999 , 2004a) have been published. However, none of these reviews includes recent advances, and none has treated all these techniques together. An early review by Lichtenthaler et al. (1998) considered reflectance and fluorescence, and these two techniques have been recently evaluated together with the focus of their potential for measuring terrestrial photosynthesis from space (Grace et al., 2007 ). Chaerle et al. ( 2007a ) have reviewed the usefulness of combined thermal and Chl-F imaging for monitoring and screening plant populations. All three types of techniques were considered together in a review by Cifre et al. (2005), but these authors focused only on their specific use in irrigation scheduling in grapevines. Meroni et al. (2009) have reviewed remote sensing of solar-induced Chl-F, whereas several recent reviews have focused on light-use efficiency and PRI (Coops et al., 2010; Hilker et al., 2010; Garbulsky et al., 2011). Only a recent review has considered together the scientific and technical challenges in remote sensing of both plant reflectance and fluorescence (Malenovsky et al., 2009).

Introduction

Although the general metabolic schemes of photosynthetic and respiratory pathways are well known when considered as separate entities, their interactions are one of the conundrums of plant photosynthetic biology. However, such interactions are the cornerstone for nitrogen assimilation by leaves, simply because carbon assimilation produces organic materials (carbohydrates) that are converted to nitrogen acceptors by respiration. Unsurprisingly then, intense efforts are currently devoted to elucidate the metabolic basis of reciprocal influences of photosynthesis and respiration, with the optimisation of nitrogen assimilation for a better yield of crop plants as an ultimate goal (Lawlor, 2002). From a basic science point of view, the interactions between respiration and photosynthesis have been a matter of interest for more than a century. The very first experiments were reported by Pizon (1902): ‘The respiration rate appears to be decreased by light. In fact, the quantity of CO2 evolved by button mushrooms (Agaricus bisporus) in the light is always smaller than that evolved in darkness. It is generally the same for green plants; however, difficulties remain to precisely assess the difference between respiratory rates in the light and in the dark, because chl.-dependent assimilation occurs in the light and one does not know how to accurately separate both phenomena.’

As emphasised by Pizon (1902), one major difficulty stems from the limited technical possibilities to measure the rate of day respiration (CO2 evolution in the light produced by respiration, as opposed to night respiration, i.e., the CO2 evolution in darkness or Rn), and importantly, from the impossibility to isolate and purify such respired CO2 molecules for example, 14C or 13C isotopic analyses. Several techniques (gas-exchange or 12C/13C techniques) have been developed over nearly 30 years to overcome this difficulty, and they are summarised below. As such, it is currently believed that photosynthesis is accompanied by lower respiration rates (inhibition of day respiration). Molecular techniques have also provided the evidence that the respiratory metabolism changes somewhat during photosynthesis in illuminated leaves, and this correlates with the operation of nitrogen and redox metabolism. At the whole-plant level, respiration is responsible for major carbon losses, but there is now a large body of evidence showing that plant growth and development is also sustained by respiration. Therefore, respiration is often viewed as beneficial for plant carbon gain at both leaf and plant levels.