3. Light

3.2.1 Avoidance of Light Stress and Permanent or Dynamic Acclimation

Lack of light, interspersed with high light intensity of short-term sunflecks, combined with a change in the spectral composition of the radiation (Fig. 3.2), is a major stressor of the vegetation on the forest floor and, less dramatically, in the shade crown of a tree (Chap. 9). As the leaves in the canopy absorb mainly blue and red light, green light is enriched in the spectral composition of the subcanopy space (“green shade”). Not visible to the human eye is another change in the spectrum of sunlight by passage through the forest canopy: the dramatic decrease in the red to far-red ratio, as the leaves absorb red and blue light but transmit and reflect far-red light (Chap. 9).

Responses to low light are obvious at the forest edges where subcanopy plants (e.g. ferns and other herbaceous species) show a pronounced phototropic reaction—that is, they grow towards the higher radiation intensity outside the forest. Plant life forms that avoid the attenuated light climate under the canopy of a forest are functional types such as epiphytes and lianas; both are light parasites that use trees as support to reach more favourable light conditions (Chap. 9).

On the other hand, a surplus of light intensity can also cause problems, as photosynthetic pigments cannot avoid energy absorption, which may overstrain the photosynthetic capacity of the chloroplasts and produce reactive oxygen species (ROS) (Chap. 2, Sect. 2.​2.​3). Leaves often avoid excessive irradiation by adopting a parallel position to the incident light. The hanging leaves of Eucalyptus are well known from the “shadeless forests” in Australia. Likewise, the upright position of the leaves of the characteristic giant rosette plants of the tropical high mountains (Fig. 4.​25) has been described as a mechanism for avoiding direct solar radiation.

The position of the leaf lamina of many plants varies during the daily light period, changing the angle and intensity of the incoming radiation. Such leaves often have joints between the petiole and the lamina, known as pulvini (Fabaceae, Oxalidaceae), which enable fast changes in the position of the entire leaves or at least the pinnules (Chap. 9). The reaction of the North American wood sorrel (Oxalis oregano) to short-term high-light stress from a sunfleck is shown in Fig. 3.3.

Dynamic acclimation to the light intensity can be observed at the cellular level too. In many algae and mosses, but also in vascular plants, chloroplasts can change their position in the cell. They accumulate on the light-exposed surfaces in the case of low light and at the light-parallel cell boundaries when the radiation becomes too strong (Fig. 3.4). Such readily reversible intracellular reactions are mediated through the blue light receptor phototropin (Wada 2013).

Besides such short-term acclimations (with a reaction time in minutes), development of leaves responds to the light environment by the so-called shade avoidance syndrome and the formation of sun and shade leaves. The shade avoidance syndrome is part of the morphogenetic reaction of seedlings when they are grown under low light intensities caused, for instance, by competing neighbours. Owing mostly to the identification of the main photoreceptors in the model plant Arabidopsis thaliana and the availability of respective mutants, knowledge on the molecular regulation of plant growth and development by light is rapidly accumulating (Casal 2013; Lau and Deng 2012; Liu et al. 2011; Lau and Deng 2012; Kami et al. 2010). Because a separation of acclimation and development is principally difficult here, we focus on the reactions triggered by light stress—that is, by either insufficient or supraoptimal light intensities.

The typical shade avoidance reaction in A. thaliana can serve as an example illustrating the integration of light cues and growth at the molecular level. Seedlings show an inhibition of lamina expansion, while petiole elongation and stem growth are enhanced. Phytochromes (PhyB) and cryptochromes (Cry1 and Cry2), as photoreceptors, have been demonstrated to play specific roles in these differential growth responses. Phytochromes especially are ideally suited to convey information on shade occurrence because of their switching between red and far-red absorption and the pronounced difference in the ratio of these two light qualities between sunlight and shade light. Low red to far-red ratios typical of shade light reduce the levels of active PhyB. Because PhyB inactivates a class of transcription factors named phytochrome-interacting factors (PIFs), their activity increases under shade light conditions. PIFs have been demonstrated to induce growth responses—for instance, through the activation of genes involved in cell wall biosynthesis. In addition, PIFs influence phytohormone signalling. They enhance auxin concentrations through the activation of auxin biosynthesis. Promotion of shade avoidance reactions by the other growth hormones gibberellic acid and brassinosteroids can be attributed to a positive effect on PIF abundance. The function of cryptochromes in shade avoidance is associated with degradation of the constitutive photomorphogenesis protein 1 (COP1) (Fig. 3.22), which is jointly triggered by cryptochromes and phytochromes, albeit in an unknown fashion different from photomorphogenesis (Casal 2013).

The shade avoidance reaction as observed under low red to far-red illumination must be attenuated when exposure to this light climate is prolonged and also when the leaf reaches into a space with sufficient light intensity. Similarly, sunflecks reduce the shade avoidance response. An autoregulatory mechanism involving transcription factors such as HY5 (Figs. 3.21 and 3.22) has been described that helps to avoid exaggerated shade avoidance responses (Jiao et al. 2007).

In contrast to trees, herbs are amenable to experimental approaches aimed at understanding the formation of physiologically and anatomically different types of leaves (Fig. 3.5). The signals indicating a high-light climate are perceived by the mature leaves, which trigger the respective development of the young leaves in the apical shoot meristem via long-distance signalling. Interestingly, the ambient CO ₂ concentration has effects on the formation of sun and shade leaves similar to those of the light intensity. High CO₂ leads to the formation of sun leaves (Prior et al. 2004; Pritchard et al. 1999).

From experiments such as those shown in Fig. 3.5 it was concluded that, at least in dicots, systemic signalling (Chap. 2, Sect. 2.​2.​4) depending on the light environment influences the development of new foliage and thus triggers acclimation to the actual light quality and intensity. Long-distance signalling from mature to developing leaves is also known from herbivore and pathogen attacks (Chap. 8), and most likely a multitude of signals is involved in mediating different types of responses. Acclimation of the photosynthetic apparatus appears to be under the control of the redox state. Thus, redox signalling may also be part of the long-distance conveyance of biochemical information. Also, the concentrations of photosynthates may act as an indirect cue for the light climate of mature leaves. However, since the formation of sun and shade leaves is a highly complex syndrome, involvement of various further signals can be expected.

At the cellular level the epidermal cells, in particular, contribute to the protection of the mesophyll cells against high-light stress. A felt-like layer of dense hairs, which reflects incident light, is found on young leaves of many plant species. Although it can be kept for permanent protection, it mostly disappears during maturation of the leaves (Chap. 9). Protective pigments, which absorb predominantly shortwave radiation, are also found in the epidermis of young leaves. A typical example is the so-called juvenile anthocyanin (Fig. 3.25), which protects not yet fully green leaves during the development of the photosynthetic apparatus. Later, protection is conferred mostly by carotenoids in the antennae. This is apparent from the disappearance of the pigments in the epidermis.

3.2.1.1 Ultrastructural Acclimation to the Light Environment

Chloroplasts of sun and shade leaves differ greatly with respect to their thylakoid systems (Fig. 3.6). Chloroplasts of sun leaves possess only thin grana, while chloroplasts of shade leaves show large grana stacks. The importance of the size of the thylakoid system in a chloroplast becomes obvious when the molecular structure of the thylakoid membranes and the functions of the photosynthetic protein complexes are considered. As photosystem II (PS II) has a larger antenna than photosystem I (PS I), over half of the total chlorophyll a, almost all of the chlorophyll b and most of the carotenoids (β-carotene and xanthophylls) are associated with PS II. The largest part of a cross-section through a photosystem consists of its antennae. PS II and its antennae are located in the so-called appressed regions—the contact zones of the stacked thylakoids. The number of antenna complexes—especially the outer or mobile antennae, which feed excitation energy to the photosynthetic reaction centre—is variable. A shade chloroplast contains a very large number of light-harvesting systems relative to the reaction centres, owing to the large thylakoid stacks and big antennae. This corresponds to a lower chlorophyll a to chlorophyll b ratio, indicating a higher proportion of the light-harvesting chlorophyll b–containing antennae around photosystem II (Table 3.1) (Kitajima and Hogan 2003). Chloroplasts of sun leaves, in contrast, contain smaller antennae and thus the ratio of antennae to reaction centres is smaller. The different organisation of the thylakoid systems of sun leaf and shade leaf chloroplasts can be considered a physiological acclimation to the ambient light environment, as both combinations allow optimal utilisation of the incident light. Such differences are observed not only in sun and shade leaves but also between the upper and lower mesophyll cells of dorsiventral leaves. Chloroplasts of the better-illuminated upper side appear as sun leaf chloroplasts, while those of the lower side (usually the spongy parenchyma) show characteristics of shade leaf chloroplasts. As the thylakoid system of a chloroplast is a dynamic substructure, fixing a leaf upside down accordingly results in a reorganisation of the thylakoid systems of the mesophyll cells.

The range of light energies known to be used for photosynthesis is huge. A champion of using low photosynthetic light intensities is a green sulphur bacterium (Chlorobium phaeobacteroides) from the Black Sea, collected from an 80-m depth, where the light intensity is 3–10 nmol quanta m⁻² s⁻¹. Thus, a single bacterium receives no more than 300 photons per second, whereas 10¹⁶ photons per second impinge on a medium-sized leaf of a terrestrial plant. Owing to high concentrations of light-harvesting pigments and very low maintenance energy requirements, the sulphur bacterium can survive but takes years to double (Overmann et al. 1992). In spite of its acclimation to the low-light environment, the efficiency of energy transfer in its photosynthetic apparatus (chlorosomes) is no more than about 60% of the absorbed radiation. Even fast-growing terrestrial plants with optimised photosynthetic membranes use less than 50% of the absorbed photosynthetic active radiation for photosynthetic CO₂ assimilation (Scholes et al. 2011) (Fig. 3.7).

On average, a dorsiventral leaf (e.g. of spinach) absorbs about 85% of the incident light between 400 and 700 nm. This is mainly due to extension of the light path in the leaf by scattering; depending on the epicuticular fine structure, up to 10% is reflected and the remaining (~5%) is transmitted (Munns et al. 2010) (Chap. 9).

3.2.2 Overexcitation and Damage to Photosynthetic Membranes

Radicals generated by overexcitation of PS II (Chap. 2, Sect. 2.​2) result first in photoinhibition (destruction of the D1 protein of the affected reaction centre). Prolonged photoinhibition leads to greater damage and finally to the destruction of the photosynthetic membranes. The potential for radical formation and photodamage is even enhanced by the oxygen-rich micro-environment of the chloroplast and the strong oxidising conditions needed for the crucial reaction of photosynthesis—the oxidation of water.

3.2.3 Flexible Acclimation to Changes in Light Intensity

The photosynthetic apparatus of higher plants serves two seemingly opposing functions: (a) harvesting of light and transfer of the excitation energy to the reaction centre; and (b) dissipation of excessively absorbed light harmlessly as heat in order to prevent photodestruction of the thylakoids. Not only can the ratio of the antennae to the reaction centres vary but also the dimensions of the antenna complexes can change in response to the light intensity (Fig. 3.8).

The so-called major antenna of PS II, LHC II (light-harvesting complex II, usually organised as trimeric complexes), together with the minor peripheral light-harvesting complexes CP26 and CP29 (chlorophyll proteins, named after their molecular weights in kDa), can dissociate from the core antenna complexes, which are also chlorophyll-containing proteins (CP43 and CP47) surrounding the reaction centre of PS II. When the energy pressure on the reaction centre is high and PS II is overexcited relative to PS I, the so-called state transition occurs (Fig. 3.8). The plastoquinone pool becomes over-reduced. This activates protein kinases that phosphorylate threonine residues in the peripheral antenna proteins. Phosphorylation leads to an accumulation of negative charges and a dissociation of the peripheral antennae from the core antennae. At the same time, the connection of the appressed regions loosens, allowing lateral movement of the peripheral antennae and thus a diminution of the PS II supercomplex (Minagawa 2013). Simultaneously, the extent (as well as the efficiency) of light harvesting of PS I increases, because part of the peripheral antennae of PS II can associate with PS I, thereby balancing the excitation of both photosystems (Fig. 3.9). Such a balance is essential for optimal utilisation of the light energy and is likewise important for the avoidance of PS II overexcitation (over-reduction of the pools of its redox compounds such as Q _B).

Even in the balanced electron flow, light intensity frequently exceeds the capacity of its utilisation for photosynthesis. According to theoretical considerations, 8 mole quanta are required for assimilation of 1 mole of CO₂. However, because of the way in which ATP synthesis is coupled to the linear electron flow, slightly more quanta are necessary. The highest measured quantum efficiency (lowest quantum requirement) is 9.4 mole quanta per mole of assimilated CO₂. For such measurements, the light intensity must be limiting, as is the case in the linear range of the light response curve (Fig. 3.10, curve a).

When the light response curve of CO₂ uptake deviates from the linear relationship (Chap. 12), more light is absorbed than can be used for photosynthesis and the plant is confronted with the need for energy dissipation. At the light intensity of a sunny day (800–1000 μmol quanta m⁻² s⁻¹), already 500–600 μmol quanta m⁻² s⁻¹ are in excess. The problem of de-energisation of the photosynthetic membranes is aggravated when water shortage necessitates closure of stomata and concomitant lowering of the intercellular CO₂ concentration. Similarly, on a bright day in winter, stress arises when the low ambient temperatures greatly decelerate metabolism and metabolite fluxes while high radiation intensities impinge on the leaves. Plants have evolved several mechanisms to cope with this challenge.

In Fig. 3.10, curve d shows the excessive photon flux density that needs to be dissipated. Less than 5% of the absorbed energy can be dissipated as chlorophyll a fluorescence (Fig. 3.9), which is complementary to the portion that can be utilised for photochemistry (photosynthesis). Because of this relation, photosynthesis can be followed indirectly by measurement of chlorophyll a fluorescence. This is illustrated in Fig. 3.11.

The intensity of fluorescence is composed of a small contribution from the antennae (termed F ₀) and a major component from chlorophyll a of the reaction centre. The latter is termed variable fluorescence (F _V). When the photosystem is completely reduced—that is, when there is no acceptor of electrons available—F _V is maximal (F _max = F ₀ + F _V). A return of F _V to the ground state (F ₀) takes about 30 s. A series of subsequent saturating light flashes decreases F _m to F _m′ because of a quench of the fluorescence of the fully reduced (“closed”) reaction centres. This quench is again composed of several components: q _p is the portion of fluorescence that is quenched by photochemistry (i.e. photosynthesis); q _E is the so-called non-photochemical quenching (NPQ) or energy-dependent quenching, which depends on the trans-thylakoid pH gradient, on the concentrations of the xanthophyll zeaxanthin and on the antenna-associated protein PsbS. NPQ is the major mechanism of photoprotection. Excess light energy absorbed by the antennae of PS II is thermally dissipated. As Fig. 3.11 shows, this quench increases with increasing saturation/reduction of PS II.

The mechanism of NPQ is complicated insofar that the same ensemble of compounds has to mediate antagonistic reactions—namely, feeding of excitation energy to the reaction centre (of PS II) as well as dissipation of excess light energy by de-energisation of excited chlorophyll molecules. Conformational changes by protonation of the involved proteins, especially of PsbS by an acidic thylakoid lumen pH, result in switching from excitation to dissipation mode (Ahn et al. 2008; Correa-Galvis et al. 2016; Fan et al. 2015). The dissipative conformation is stabilised by zeaxanthin, which is a product of the xanthophyll cycle (Fig. 3.12). In high light and correspondingly low thylakoid lumen pH, violaxanthin de-epoxidase is activated, which catalyses the formation of zeaxanthin from violaxanthin. A model has been proposed that integrates the fast component of NPQ via protonation of PsbS and the slow component, the formation of zeaxanthin by the de-epoxidation of violaxanthin and subsequent association with PsbS (Zaks et al. 2012, Fig. 3.13). The fast component may be restricted to the already detached antennae, whereas the slow component appears to happen in the core antennae, including the minor chlorophyll proteins, and thus protects PS II from overexcitation (Holzwarth et al. 2009). The quantitatively dominant xanthophyll of the thylakoid membranes, lutein, can react in a way similar to zeaxanthin (Li et al. 2009). A lutein-5,6 epoxide cycle has been reported, which is driven by light-activated violaxanthin de-epoxidase and zeaxanthin epoxidase under low light (Matsubara et al. 2008). The physiological importance of the xanthophyll cycles is underlined by the increase and decrease in the concentrations of their components upon transfer of a plant from low light to high light, and vice versa (Fig. 3.14). Also, one of the first common garden experiments with A. thaliana wild-type and mutant plants demonstrated that plants with defects in NPQ suffer a significant loss of fitness (determined as seed yield) under naturally fluctuating light conditions (Kühlheim et al. 2002).

At the metabolic level of the Calvin cycle, even under conditions of closed stomata, a possibility to avoid or at least reduce overexcitation arises from the oxidative photosynthetic carbon cycle (photorespiration) which, under low CO₂ (due to decreased conductance of the stomata) and high light, releases CO₂ from glycine decarboxylation. This internal CO₂ keeps the photosynthetic electron flow running through consumption of NADPH and ATP in the Calvin cycle (for the reactions and compartmentation of the oxidative carbon cycle, see plant biochemistry textbooks). Likewise, photosynthetic reduction of nitrite or sulphate and the subsequent formation of amino acids require these photosynthetic primary products. However, the rates of the latter pathways are comparatively small. Oxygen may also be reduced photosynthetically in the so-called Mehler reaction (Chap. 2, Sect. 2.​2), giving rise to ROS, which can be detoxified by a sequence of reactions, finally resulting in the consumption of NADPH (the “water–water cycle” or pseudocyclic photosynthetic electron flow) (Fig. 3.15).

Box 3.1: A Day in the Life of a Tree Leaf: Dynamic Acclimation of Leaf Performance to Short-Term Environmental Changes

The effect of sunflecks on the photosynthetic performance of leaves (e.g. of a tree crown) has been discussed controversially with respect to the question as to whether a frequent change in the light intensity enhances or decreases their photosynthetic efficiency. The basis of such considerations is the observation that once a leaf is in a photosynthetically active state, the increase in the rate of photosynthesis is fast (occurring in a few seconds), while the return to the “shade state” is in the range of minutes, resulting in overall elevated efficiency (Fig. 3.16c). On the other hand, such a consideration must also take note of the nonlinear quantum efficiency of varying light intensities, which is described by the light response curve of net CO₂ uptake (Figs. 3.10 and 3.16a). Sunflecks from direct sunlight commonly surpass the range of the linear or nearly linear relation between light intensity and photosynthetic CO₂ uptake. In the examples shown in Figs. 3.16a and 3.17a for the tropical gymnosperm Podocarpus falcatus, the increase in photosynthetic CO₂ uptake was minimal beyond a photosynthetically active radiation (PAR) intensity of about 400 μmol quanta m⁻² s⁻¹. The sunflecks, however, reached up to 1500 μmol quanta m⁻² s⁻¹ (Fig. 3.17a). Therefore, the sunflecks were of lower photosynthetic quantum efficiency than low light. When the same amount of PAR was supplied to a Podocarpus leaf over the same time period as continuous radiation of low intensity (Fig. 3.16b) or as artificial sunflecks (termed lightflecks; Fig. 3.16c), the photosynthetic gain was obviously higher under continuous low light. Under natural conditions, not only the light intensity varies (Fig. 3.17) but also the conductivity (g _s) of the stomata responding to the water status of the leaves (Chaps. 10 and 12). When stomatal conductivity is low, as it generally is in the afternoon, the photosynthetic efficiency of sunflecks is even lower because of a low internal CO₂ concentration. This in turn indicates the importance of energy dissipation by the chloroplasts. The actual quantum efficiency of CO₂ assimilation over an entire day was only 72% of the (theoretical) quantum efficiency of the same amount of quanta administered over the same time period, when provided as continuous low light (Table 3.2). This difference is, of course, not a constant. However, it indicates the extent to which the real light climate is less effective than artificial illumination. Continuous artificial low light mimics, to some extent, the diffuse radiation in the shade of a tree canopy which, for an entire forest canopy, is photosynthetically more efficient than direct irradiation (Mercado et al. 2009). In comparison with the mentioned minimal quantum requirement for photosynthetic CO₂ assimilation of 9.2 μmol quanta per μmol CO₂, the data in Table 3.2 suggest a sixfold lower quantum efficiency (54 mole quanta per mole of CO₂). Under adverse environmental conditions the apparent quantum efficiency can further decrease tremendously—for example, when stomata are closed under drought and merely 1–2% of the incident visible light can be used for photosynthesis (Osmond et al. 1997). It is important to note that quantum efficiency has a different meaning in the ecological context than it does in the photosynthetic light reaction, where only those quanta that conduct photochemistry are counted. The difference is due to the energy of a high proportion of absorbed quanta being dissipated as heat under natural conditions.

Table 3.2

Effect of sunflecks on the photosynthetic gain of a tree. Photosynthetic efficiency of the subcanopy light climate compared with virtual constant illumination of the same PAR magnitude applied over the same time period to leaves of Podocarpus falcatus (Modified from Strobl et al. (2011))

Daily sum of PAR [mol m−2 day−1]	Calculated PARav over the day (μmol m−2 s−1)	Theoretical daily CO2 net uptake on the basis of PARav (mmol m−2 day−1)	Measured daily CO2 net uptake (mmol m−2 day−1)	Apparent efficiency of actual PAR (%)
1.5	50.5	38.3	27.7	72

PAR photosynthetically active radiation, PAR _av average PAR intensity

3.2.4 Continuous Light

In greenhouses, plants are frequently cultivated under continuous light for enhancement of biomass production. On the other hand, continuous light can induce severe injury in many plants. Plants sensitive to continuous light (e.g. eggplant, peanut, some cultivars of potato and many cultivars of tomato, but also some lichens and mosses) react with lower rates of photosynthesis, leaf chlorosis and necrosis (Velez-Ramirez et al. 2011). Several reasons have been identified for that damage. Some of it results from a disturbance in photosynthetic carbohydrate metabolism. Assimilatory starch accumulates during the daily light period between the grana of the chloroplasts. Suppression of nocturnal starch degradation leads to continuous deposition of starch and finally destruction of the chloroplast ultrastructure. Enhanced formation of radicals and ROS are other reasons, in particular when the applied light contains a high proportion of blue light. Usually the natural day/night cycle is accompanied by an oscillation in the temperature. Continuous light in combination with a constant temperature can disturb the circadian clock and affect the development of the plant. Is continuous light not a natural phenomenon of the polar summer beyond the polar circles, one might ask? By no means! Even in the polar summer, the light intensity (and light spectral distribution) oscillates in the daily rhythm concomitantly with the temperature. Thus, artificial continuous light (and temperature) provided in a greenhouse represents an environment without an analogy in nature. It is therefore maybe more surprising that some plants can grow well in continuous light than that many plants cannot.

3.2.5 Light Triggers Plant Adaptation and Acclimation to the Environment

3.2.5.1 Photoreceptors

Because of its role as an energy source, light is arguably the most important environmental factor for a plant. Consequently, perception of light and translation of the respective signals control many aspects of plant development and are crucial for adaptation to diverse habitats. The sensing of light conditions is enabled by a range of receptors, which are specific for particular wavelengths of the visible spectrum (Fig. 3.18). Phytochromes are receptors for red and far-red light, cryptochromes and phototropins are receptors for blue light. The photoreceptors for red and blue light principally consist of a protein and a chromophore. The chromophores are molecules absorbing light of the respective wavelengths (for example, flavin adenine dinucleotide (FAD) and tetrapyrroles). During evolution they have been recruited for a wide range of biological functions. Upon photon absorption the chromophores undergo conformational changes that are transmitted to the protein and thereby change its activity. For UV light a special photoreceptor has recently been identified (UVR8) which, because of the UV absorption by aromatic amino acid residues, does not require a chromophore.

Phytochromes

Phytochromes are proteins with an open-chain tetrapyrrole as a chromophore that changes its configuration upon absorption of red or far-red light. The effective ratio of red to far-red, ζ, refers to the broad spectral peaks of phytochrome in the red and far-red and can be calculated by Eq. 3.1 (Chelle et al. 2007):

(3.1)

where E _R and E _FR are the spectral irradiances from the sun and the sky and the complex radiative transfer reactions within the canopy. E _R comprises the radiation between 655 and 665 nm and accordingly E _FR comprises that between 725 and 735 nm. Whereas ζ of the sunlight is around 1.2, it decreases in the understorey space to less than 0.2. Reactions that are triggered by (red) light (e.g. light-dependent seed germination) thus do not take place in the shade of a dense canopy. The situation changes in forest gaps where, because of the direct irradiation, ζ is high and seeds of pioneers can germinate rapidly (Chap. 2, Sect. 2.​4). In contrast, the time span of sunflecks is too short to initiate red light–triggered morphogenic reactions, because the activation of phytochrome is reversible by a rapid return to low ζ.

The scheme in Fig. 3.19 shows two phytochromes (A and B). Phytochrome B represents several phytochromes (B through E), which react in the same way and have been identified from several plant sources. While the interconversion of the physiologically inactive form (“P_r” for P_red) to the active form (“P_fr” for P_far-red) by respective irradiation follows the same mechanism in all phytochromes, the inactivation mechanisms for P_fr differ. PhyA_fr inhibits its own synthesis, and irradiation with red light as well as with blue light triggers its degradation. Synthesis and degradation play a secondary role in PhyB biochemistry as compared with photoconversion and slow reversion in the dark (Fig. 3.19). Note that photoconversion of phytochromes is associated with transport into (P_fr) and out of (P_r) the nucleus. The existence of a photoreceptor in two different states with distinct absorption maxima allows the monitoring of spectral quality (similar to colour vision in animals). Sunlight comprises both red and far-red light. Hence, the ratio of red to far-red light is crucial for the concentration of P_fr and the formation of a cellular signal.

Cryptochromes

Three cryptochromes have been found in A. thaliana, two acting primarily in the nucleus and one (Cry3) in mitochondria and chloroplasts. Cry3 is a photolyase with potentially some cryptochrome activity too (Liu et al. 2011). Cryptochrome 1 is the photoreceptor for high blue light intensity, while cryptochrome 2 is a sensitive blue light receptor that reacts at low fluence rates. The photoactive domains of the cryptochromes are homologous to those of the photolyases (N-terminal photolyase-homologous region (PHR)). However, because of their C-terminal extensions, cryptochromes do not exhibit photolyase activity (Fig. 3.20) but instead act as kinases. The photochemical mechanism(s) of cryptochrome light activation are not yet completely understood and the mechanisms of photoactivation and inactivation of both cryptochromes may differ to some extent (Li et al. 2011; Liu et al. 2011). Binding of the chromophores (5,10-methenyltetrahydrofolate (MTHF)) and FAD to the protein is non-covalent and the formation of an FAD radical anion (by electron transfer from MTHF) and a reduced FAD (FADH) radical (by subsequent proton transfer from the protein) appears obligatory in both cryptochromes. For activity, A. thaliana cryptochromes have to be phosphorylated. Upon additional (auto)phosphorylation after exposure to blue light, Cry2 (but not Cry1) is ubiquitinated and degraded. This mode of action resembles the situation in the phytochrome systems where PhyA is readily degraded, while PhyB is not (Liu et al. 2011).

Phototropins

Phototropins (Phot 1 and Phot 2 in A. thaliana) are blue light receptors of the plasma membrane with two flavin mononucleotides (FMNs) as chromophores. In darkness, their C-terminal protein kinase domain is sterically inhibited. Absorption of blue light changes the protein conformation, leads to dissociation from the plasma membrane and unlocks the kinase activity. In the activated state, the FMN becomes covalently bound to a cysteine residue of the protein chain. This state of the protein is unstable and is readily reversed in the dark, whereupon the covalent bond breaks up. Along with cryptochromes and phytochromes, phototropins allow plants to respond to their light environment. For instance, they are important for the opening of stomata and mediate phototropic responses, e.g. the movements of chloroplasts (Fig. 3.4). Phot 1 is required for blue light–mediated transcript destabilisation of specific messenger RNAs (mRNAs) in the cell. For detailed chemical structures of the photoreceptors and mechanisms of gene regulation by light, see plant biochemistry textbooks (e.g. Buchanan et al. 2015).

3.2.5.2 Signal Transduction: The Signalling Centre COP1

Growth of plants in the dark, known as etiolation or skotomorphogenesis, requires suppression of photomorphogenesis, which is initiated by illumination. Activation of the above described photoreceptors - phytochromes and cryptochromes in particular - by irradiation triggers signal transduction cascades, which converge at a protein termed COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1). COP1 is a central switch which, because of its complex structure, can interact with several other proteins. In the dark, COP1—by its function as an E3 ubiquitin ligase—targets photomorphogenesis-promoting transcription factors for degradation by the ubiquitin-proteasome system, thus preventing photomorphogenesis. One of these proteins is the transcription factor ELONGATED HYPOCOTYL 5 (HY5) which, together with others, is responsible for the onset of photomorphogenesis (e.g. the inhibition of hypocotyl elongation growth). In the dark, it is degraded in the proteasome, giving rise to the well-known etiolated hypocotyls. Light-activated photoreceptors inactivate COP1 by mediating its export from the nucleus, thereby suspending its activity against transcription factors that are located in the nucleus. For a further understanding of the interaction of COP1 with the photoreceptors, it is necessary to introduce another protein, SPA1 (SUPPRESSOR OF PHY A 1), which forms a complex with COP1 to enable its association with the E3 ligase core complex; this finally ubiquitinates the proteins, targeting them for degradation (Fig. 3.21a). In the light, activated Cry1 and 2 interact with SPA1, dissociating it from COP1, which can now be exported from the nucleus (Fig. 3.21b). Further targets of the COP1 E3 ubiquitin ligase are the phytochromes—in particular PhyA_fr and cryptochrome 2.

While COP1 is a negative regulator of photomorphogenesis, it promotes UV-B-triggered reactions of plants, hence acting as a positive regulator (Fig. 3.22). To understand this difference, it is important to note that in skotomorphogenesis, COP1 acts as a homodimer, and also the COP1–SPA1 complex is dimeric. Dissociation of the COP1–SPA1 complex by sequestration of SPA1 through interaction with cryptochrome also monomerises the COP1 homodimer. In the cytosol, monomeric COP1 can bind to the monomeric activated UVR8 receptor and the heterodimer migrates back into the nucleus, where it activates HY5 and other transcription factors and effectors (Chap. 3, Sect. 3.4).

3.3.1 Ranges of Ultraviolet Radiation and Biological Activity

The spectrum of solar ultraviolet light is continuous but is commonly divided into three wavelength bands: UV-C (200–290 nm), UV-B (290–320 nm) and UV-A (320–400 nm). UV-C is the most energetic of the three and is known as “germicidal UV” because of its potency against microorganisms (Yin et al. 2016). It is used to disinfect fresh fruit and vegetables to preserve their quality. However, since it is effectively absorbed by oxygen and ozone in the stratosphere, only a very small fraction reaches the Earth’s surface. UV-A, on the other hand, is not attenuated by atmospheric ozone, and this less damaging type of radiation plays an important role in plant photomorphogenesis. Although a sizeable amount of UV-B is absorbed by atmospheric ozone, its impact on life on our planet is considerable.

The development of the ozone hole—that is, the decrease in stratospheric ozone concentrations due to ozone decomposition by reaction with anthropogenic gases such as halogenated hydrocarbons or nitrogen oxides (IPCC/TEAP Special Report on Ozone and Climate 2005) is therefore observed with much concern. While the seasonally fluctuating ozone hole is particularly pronounced in the polar and subpolar regions, the UV radiation emitted by the sun crosses the atmosphere in regions of high geographical latitude at an angle and thus encounters a significantly “thicker” ozone layer than at the equator, where it takes the shortest path through the atmosphere. Therefore, in spite of the high latitudinal ozone hole, the UV radiation is high in the tropics and relatively low in the polar regions. The well-known altitudinal increase in UV radiation is caused by an attenuation of the tropospheric ozone layer, concomitant with a decrease in the intensity of haze.

UV-B radiation can damage cells and thus is dangerous to organisms (Table 3.3). In particular, plants—as sessile organisms—have developed mechanisms to cope with the ubiquitous and inescapable natural flux density of UV radiation through repair mechanisms for damaged cellular components (such as DNA). Protective measures are accumulation of UV-absorbing pigments in the epidermal layers, thick layers of hairs (Holmes and Keiller 2002; Manetas 2003) and cuticular waxes (Barnes et al. 1996). Generally, plants show adaptation to the UV-B load of their habitat. Plants growing along a latitudinal or an elevation gradient exhibit increased UV-B tolerance (Robberecht et al. 1980, Fig. 3.23). Problems caused by UV may arise for crop cultivars that are not well adapted to the natural UV-B stress occurring at their sites of cultivation, or for mobile organisms such as plankton. Phytoplankton, especially of the cold oceans, appear to be very sensitive to UV-B. In spite of the shallow depth to which UV light penetrates in a body of water (Fig. 3.24), significant reductions in phytoplankton biomass have repeatedly been observed as a consequence of the ozone hole. Likewise, decreases in yields have been reported for UV-sensitive crop cultivars (e.g. of maize and soybeans (Rius et al. 2016)). However, although statistically significant, these reductions were relatively small (usually <10%).

Table 3.3

Physiological effects of increased ultraviolet (UV)-B radiation. (Modified from Jansen et al. (1998))

Damage
DNA damage	Dimerisation of thymine; strand breaks
Biomembranes	Lipid peroxidation
Photosynthetic apparatus	Inactivation of photosystem II; acceleration of D1 turnover; damage to thylakoid membranes; bleaching of pigments; decrease in the activity of photosynthetic enzymes (particularly RubisCO); inactivation of photosynthetic genes
Phytohormones	Photo-oxidation of auxin
UV avoidance
Activation of secondary metabolism	Activation of expression of the key genes of phenylpropanoid metabolism and accumulation of flavonoids; accumulation of alkaloids, waxes and polyamines
Production of radical scavengers	Increased capacity of the anti-oxidative system (ascorbate peroxidase, superoxide dismutase, glutathione reductase and others, e.g. mycosporines)

RubisCO ribulose-1,5-bisphosphate carboxylase/oxygenase

Morphological–anatomical symptoms indicating a still tolerable UV-B stress are swollen and shortened internodes, reduced leaf expansion, curling up of leaf edges and enhanced branching of the shoot through promotion of lateral buds. Leaves tend to show succulence with a particularly thick, usually pigmented epidermis and low density of stomata. Tolerated UV stress often results in accumulation of vitamin C and soluble sugars in fruits as radical scavengers, thereby also increasing the quality of food. However, the transition from UV-B-triggered “normal morphogenetic reactions” to those that have to be considered as stress responses is not clearly apparent, inasmuch as other natural stresses such as drought, nutrient deficiency or low temperature can interact with UV-B effects at the molecular level. Nevertheless, differentiation between normal development of UV-B tolerance and stress responses is clear when above-ambient levels of UV-B cause damage to DNA, proteins and membrane lipids, and inhibit protein synthesis and photosynthetic reactions. However, since UV-B also generates ROS (mainly the superoxide radical O₂ ⁻) through its impact on photosynthesis, respiration and on enzymes such as peroxidases and oxidases, the origin of UV-B triggered damage is not always obvious.

Investigation of UV-B effects on plants is not trivial, because of the omnipresence of this radiation in nature and, to a lesser extent, in artificial light sources. Many experiments have therefore applied pulses of UV-B overdoses or longer than natural exposure periods. Such treatment can easily overstretch the tolerance—for example, the capacity for repair—of the plants, causing unnatural reactions and even necroses and death. Also, different reactions have been observed, depending on whether the same total overdose of UV-B was applied either in high intensity pulses or as a slightly elevated constant flux over a long period. With the UV stress applied in short pulses, damage and repair are the dominant effects, while continuous but low-intensity stress leads to acclimation and damage avoidance.

3.3.2 Ultraviolet-B Damage and Repair Mechanisms

DNA has a broad absorption peak between 235 and 315 nm and thus is photoactivated by UV. Since the effects of UV-B radiation on DNA affect all kinds of organisms, they have been particularly well studied. Several types of UV-triggered damage are known: strand breaks and cross-linking, as well as modifications of pyrimidine bases. In plants, dimerisation of thymine—resulting in cyclobutane pyrimidine dimers (CPDs) and, to a smaller extent, in pyrimidinone dimers (known as 6–4 photoproducts)—is a common reaction to irradiation with UV-B. Less packaged DNA of mitochondria and probably also of chloroplasts is particularly sensitive. UV irradiation of yeast cells caused a 10% loss of nuclear DNA, but at the same time a 50–60% loss of mitochondrial DNA.

There are several mechanisms to repair such damage (for details, see biochemistry textbooks, e.g. Berg et al. (2015)), of which two are mentioned here: the DNA photolyase reaction, which requires blue light/UV-A; and light-independent reactions such as base excision and recombination. Repair usually takes only a few hours. Interestingly, UV-activated photolyases represent the evolutionary origin of the blue light receptor cryptochrome (Fig. 3.20). Strong UV stress leads directly to irreparable chromosome breakage and deletions resulting in the death of the organism. This is the basis for sterilisation of rooms and instruments with intense UV light.

Irrespective of the repair mechanisms, DNA damage signalling involves a UV-damaged DNA-binding protein complex (UV–DDB complex), which recognises UV-induced DNA damage and recruits proteins of the nucleotide excision repair pathway. It also induces several other UV-B responses, such as the formation of protective pigments.

In addition to UV damage to DNA, photo-oxidation of UV-absorbing pigments is known: yellowing and complete bleaching of leaves of indoor plants after abrupt transfer to the open air are frequent phenomena. Here the protective effect of the chloroplast pigments (energy dissipation) on their protein environment can be seen. Photodestruction of the thylakoid pigments leads to a significant decline in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO).

3.3.3 Avoidance of Ultraviolet-B-Induced Stress

Plants synthesise a range of compounds in order to avoid damage by UV-B radiation. The rate of accumulation in different plant types correlates well with the average UV exposure associated with the respective habitat (Fig. 3.23). Phenylpropanes, as aromatic compounds, exhibit strong absorbance of shortwave radiation and are thus able to function as effective UV filters or sunscreen pigments. Many compounds in this family of secondary plant metabolites absorb light only in the UV range and cannot be recognised as pigments by the human eye. Others, such as the anthocyanins, absorb light also in the visible range and therefore appear as pigments (Figs. 3.25 and 3.26).

Glycosylation renders phenylpropanes water soluble so they can be sequestered in the vacuoles primarily of the epidermis. In certain purple varieties such as copper beech (Fagus sylvatica), the vacuoles of the mesophyll cells contain such pigments too. They absorb in the UV spectrum (Fig. 3.26) and in the green region of the visible spectrum and thus do not interfere with the absorption of light by the chlorophylls. Protective colouration of the leaves by anthocyanins is frequently observed in unfolding young leaves (called “juvenile anthocyanin”) and again during senescence. In young leaves the additional pigments protect the developing photosynthetic machinery until the energy dissipation mechanisms are fully functional. The “autumn anthocyanin” is well known from the Indian-summer aspect of deciduous trees. Its ecological function, however, is not well understood. Most likely it protects the controlled degradation of leaf constituents—in particular, the chlorophylls—and the export of degradation products to the overwintering parts of the plant. The key enzyme in the flavonoid metabolism is chalcone synthase, whose formation is strongly induced by UV-B as well as by UV-A/blue light.

Other examples of compounds—out of the great variety of aromatic secondary metabolites—that accumulate upon UV-B exposure include 3″,6″-DCA and 3″,6″-DCI (=3″,6″-di-para-coumaroyl-astragalin and 3″,6″-di-para-coumaroyl-isoquercitrin) in pine needles and 2″,2″-di-para-coumaroylkaemperol-3α-d-arabinoside in beech leaves (Figs. 3.27 and 3.28). The non-conjugated phenylpropanes (e.g. p-coumaric acid and its derivatives) are primarily localised in the epidermal cell wall, while the conjugates are mainly sequestered in the vacuoles of the same cells. It has been calculated that the accumulation of aromatic compounds in the outer epidermis cell wall of pine needles allows only about 4% of the incoming UV-B to pass through. Diacylated flavonoids in the vacuole are able to filter out the residual UV-B, leaving the mesophyll completely unaffected. This explains the exceptional UV protection that needles of coniferous trees are known for (Fig. 3.23).

The association between concentrations of UV screens and UV exposure in the natural habitat is apparent when comparing, for instance, alpine herbs with herbs from the lowlands (Filella and Peñuelas 1999; Fig. 3.23). Moreover, because synthesis of UV-B screening phenols and phenylpropanes is stimulated by UV-B radiation, variations in their concentrations can be expected also when leaves of the same species but from locations with contrasting UV-B radiation intensities are compared. One example of such a local adaptation is mountain avens (Dryas octopetala) from the Arctic, from southern Norway and from the French Alps, where the average UV-B radiation is three times higher than in the Arctic. UV-B transmittance in the epidermis of Dryas from the French Alps was only 2.5% versus 5% in plants from Norway and 7% in Arctic plants (Nybakken et al. 2004). It was also shown that depending on the weather conditions during the year, the screening capacities of the leaf epidermis may change, indicating a dynamic acclimation response (Barnes et al. 1996).

3.3.4 Ultraviolet-B Perception and Signalling

Many transcriptional responses to increased doses of UV-B are non-specific and shared with, for instance, defence responses against pathogen attack (Stratmann 2003). The respective signalling has been associated with UV-B as a severe stressor of plant life, is triggered by DNA damage and ROS production, and involves typical plant stress hormones (Fig. 3.29). Responses result in repair and in protection by the same classes of chemical compounds, which can act either as sunscreen pigments or as components for defence (phytoalexins). In particular, induction or reinforcement of radical scavenging systems such as glutathione reductase, ascorbate peroxidase and glutathione peroxidase are very important for avoidance and alleviation of UV-triggered stress. Overall, responses to UV-B stress are dependent on signalling pathways triggered by non-specific secondary stress (Chap. 2, Sect. 2.​2), while photomorphogenic acclimation responses are more UV-B specific and are triggered by low levels of UV. It is important to note that both contribute to survival. The specific responses can—depending on the wavelength, intensity or duration—also represent acclimation and avoidance reactions. During the past decade, much progress has been made in the elucidation of signal transduction chains resulting in UV-B tolerance of plants.

Photomorphogenic signalling requires perception of radiation. While the photoreceptors for visible light contain chromophores, the UV-B photoreceptor lacks such a component. It absorbs shortwave radiation by a series of aromatic amino acid residues—in particular, 14 tryptophan residues (Trp, W). The UV-B receptor was originally described as a regulatory protein in UV-triggered signal transduction in A. thaliana (Kliebenstein et al. 2002) and was termed UV RESISTANCE LOCUS 8 (UVR8). In 2011, Rizzini et al. identified this protein as the long-sought-after UV-B photoreceptor which, by a special arrangement of tryptophan residues and positively charged arginine residues, can convert UV-B radiation into a chemical signal. Other authors contributed the detailed photochemical and biochemical mechanisms of that process (Christie et al. 2012; Wu et al. 2012). In all investigated species, from algae to higher plants, this type of UV-B photoreceptor has been found (Rizzini et al. 2011).

In the energetic ground state (e.g. in the dark), UVR8 is a doughnut-shaped homodimer whose monomers are linked by a network of salt bridges and aromatic side-chain interactions (Gardner and Correa 2012) (Fig. 3.30). Each monomer consists of 440 amino acid residues with 14 Trp residues, which are clustered at the top surface where the dimer forms. Three of them form a triad, which interacts with another tryptophan of the counterpart analogue (Fig. 3.30). Excitation of the tryptophan residues by UV-B results in the dissociation of the salt bridges and releases the monomers. The active UVR8 monomer then binds to the multifunctional COP1 protein, which is a central regulator in UV-B and visible light signalling (Oravecz et al. 2006) (Fig. 3.22). At this point the question of “normal photomorphogenesis” versus the UV stress response arises again but cannot be conclusively answered. It is, however, clear that UV-B-specific responses—whether they are contributing to the normal development of a plant or are excited by an unusual dose of UV radiation—are mediated by the UVR8–COP1 signalling pathway (Heijde and Ulm 2012). The central protein of this pathway is COP1, best known as a negative regulator or repressor of photomorphogenesis (Fig. 3.22). However, in UV-B signalling, COP1 is a positive regulator. The interaction complex of monomeric UVR8 with COP1 migrates from the cytosol into the nucleus, where it activates HY5 gene expression. The transcription factor HY5 triggers the expression of a great variety of UV-B-responsive genes. Among these are genes that encode proteins required for UV-B tolerance or protection, such as photolyases for DNA repair, and enzymes of the phenylpropanoid pathway, such as chalcone synthase, by which phenolic UV-B scavengers are produced.

3.3.5 Crosstalk Between Ultraviolet-B and Visible Light Responses

COP1 interacts with all known photoreceptors for visible and UV-B light in specific ways, thereby enabling crosstalk between the photoreceptors. The multifunctionality of the COP1 protein is based on its complex structure, which consists of three major domains: a RING finger, a coiled coil domain and a so-called WD40 (TrpAsp) repeat domain. COP1 can dimerise through the coiled coil domain. Furthermore, the photoreceptors for red/far-red (phytochromes), blue (constitutive cryptochrome effect) and UV-B (UVR8) interact with specific sites of the WD40 domain. By the versatility of the interactions of photoreceptors with the same central regulatory protein, COP1 specificity of signalling can be achieved. While, for example, the ubiqitination and degradation of the photolabile receptors Cry2 and PhyA is mediated by COP1 (Fig. 3.22), it does not impair the stability of PhyB, Cry1 and UVR8. Further regulation of COP1 activity and specificity is achieved by interaction of the coiled coil domain with proteins of the SUPPRESSOR OF PHYTOCHROME A family (SPA1–SPA4; Fig. 3.21), which are required for COP1 function in the dark and visible light but not for the UV-B response. On the other side, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 and 2 (RUP1 and RUP2, Fig. 3.30) have been identified as negative feedback regulators of UVR8 signalling (Gruber et al. 2010).