Water accounts for up to 90% of a plant’s fresh weight. Furthermore, since the experiments of Stephan Hales in the eighteenth century it has been known that a large fraction of the water taken up by plants from the soil is lost to the air, that is, it is transpired. This water loss is an inevitable consequence of the need for terrestrial plants to take up CO₂ from the atmosphere. For every molecule of CO₂ that enters a plant through stomata by diffusion, several hundred molecules of H₂O leave the plant via this same pathway. The concentration gradient across the leaf surface is much greater for water loss than for CO₂ uptake, but a membrane or other material that would allow selective passage of CO₂ has never evolved in plants. Thus, not only is water the most abundant of the resources needed by a plant for functioning and growth—a characteristic that plants share with animals—but CO₂ uptake and thereby photosynthesis require large fluxes of water through the plant, which is the reason why water availability very often limits productivity (Chap. 10). The strict correlation between CO₂ uptake and water loss is sometimes referred to as the central dilemma of plants: dying of thirst or dying of hunger?

A second distinctive feature of plant–water relations is based on a major difference in the structure of plant and animal cells. Plant cell walls can withstand considerable hydrostatic pressures and tensions. The ability to build up turgor pressure is essential for growth via cell expansion and for the rigidity of tissues not stabilised by lignified cells.

Besides temperature, precipitation is the most dominant environmental factor determining the distribution of vegetation on the global scale. Vast differences exist between plant species in the ability to grow and reproduce in water-limited habitats, estimated to represent more than 50% of the Earth’s surface area. Following a consideration of the unique properties of water, this chapter will address cellular aspects of plant–water relations such as the driving forces for water movement, the water conductivity of membranes and cellular responses to water scarcity. Plants synthesise a range of protective molecules, regulate their osmotic potentials and—most importantly—control the rate of water loss through stomata (Fig. 6.1). Also covered are the molecular mechanisms underlying growth responses to drought and the photosynthesis variants (C ₄ photosynthesis and Crassulacean Acid Metabolism (CAM)) that result in higher water use efficiency, i.e., a more favourable ratio of water loss to CO₂ fixation. Plant–water relations at the whole-plant level are described in Chap. 10.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig1_HTML.png — Fig. 6.1
A plant before (*left*) and during (*right*) drought stress. Major control points of acclimation are indicated as valves. (Modified from Maggio et al. (2006))

6.1 The Properties of Water

The biochemistry of life requires water in the liquid state and is thus dependent on the physico-chemical properties of the water molecule. These are often referred to as anomalies (Box 6.1) and result from the dipole nature of the molecule H^δ+-O^δ−-H^δ+ (Fig. 6.2a). The water molecule is bent (at an angle of around 104°) and therefore has an asymmetrical charge distribution. The dipoles produce hydrogen bonds between the individual molecules, and this guarantees a high degree of cohesion with, at the same time, low viscosity. The water molecules form flickering (mobile) clusters or aggregates, which continuously exchange individual molecules. That explains why water is in the liquid rather than the gaseous state at temperatures between 0 and 100 °C and at standard air pressure, despite the low molecular mass of the water molecule. Related consequences of the dipole nature of water are adhesion to polar surfaces such as cell walls, and capillary forces caused by the high surface tension. Adhesion, capillary forces and cohesion, as well as low viscosity, are decisive factors for the transport of water from roots to leaves (the cohesion–tension theory of water conductance; Chap. 10 and plant physiology textbooks). Another consequence of the dipole nature of the water molecule is its suitability as a solvent for polar and polarisable compounds. In addition, water exerts a structuring force in amphiphilic systems (hydrophilic–hydrophobic) giving rise to lipid micelles and contributing to the tertiary structures of proteins. Furthermore, water is a very effective heat buffer for organisms because of its relatively high heat of crystallisation (freezing avoidance) and very high heat of vaporisation (transpiration cooling). As its radiation absorption is outside the boundaries of the visible spectrum, water does not absorb visible light and thus does not interfere with photosynthesis or processes regulated by blue or red light.

Box 6.1: The Physico-chemical Properties (Anomalies) of Water

Because of their dipole nature, water molecules (Fig. 6.2a) associate via hydrogen bonds to a three-dimensional lattice (clusters, Fig. 6.2b), which is in a permanent molecular rearrangement. The physico-chemical anomalies of water (e.g. high melting and boiling temperatures in comparison with molecules such as H₂S or ethanol, Table 6.1) can be attributed to this cluster formation.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig2_HTML.png — Fig. 6.2
Dipole, clusters, hydrophobic interaction

Table 6.1

Physico-chemical anomalies of water

Compound	Molecular weight	Melting point (°C)	Boiling point (°C)
H₂O	18	0	100
H₂S	34	−86	−61
Further biologically important physical properties of water
Specific heat capacity		1 cal g⁻¹ = 4.2 J g⁻¹
Latent heat of crystallisation		333.6 J g⁻¹
Latent heat of evaporation		2441 J g⁻¹
Surface tension (at 15 °C)		73.5 g s⁻¹

The properties of water are also the reason for hydrophobic interactions. Non-polar molecules in an aqueous environment are forced into aggregates in order to minimise the energetically unfavourable interaction with water molecules. Amphiphilic molecules (i.e. molecules with polar and non-polar groups) such as phospholipids and glycolipids form ordered structures (= biomembranes) with the hydrophilic part interacting with water molecules and the hydrophobic part excluding water (Fig. 6.2c) (Larcher 2003).

6.2 Water Acquisition and Movement: Cellular Aspects

Life is so strictly dependent on water that the search for traces of extraterrestrial life (e.g. on Mars) is essentially a search for signs of water. The evolution of life on land has required key innovations allowing organisms to acquire water. In addition, most animal and plant species have to maintain a hydrated state; that is, they are homoiohydric. Only few species of vascular plants can withstand dehydration and are able to resume physiological activity after rehydration. Together with lichens and many mosses, they are referred to as poikilohydric.

The needs to take up water and to stay hydrated demand acclimations and adaptations to cope with fluctuations in water supply and, in particular, with a shortage of water supply. Many plant habitats are characterised by either temporary or permanent water scarcity. Not only does this threaten photosynthesis and the functions of all kinds of metabolic processes that take place in the aqueous environments of cells, but water is also physically important for the growth of plants. Growth is a function of cell division and cell expansion. The expansion growth of plant cells is dependent on turgor pressure, i.e. on water influx. The modulation of cell wall properties such as their extensibility controls the expansion rate, but the build-up of hydrostatic pressure is what drives the expansion.

Thus, practically every facet of terrestrial plant life is dependent on the uptake of water from the soil at sufficient rates and the tightly controlled movement of water within the plant. Water uptake by roots and water movement are dependent on a driving force—that is, pressure differences—and on the facilitation of passage through biological membranes. We consider cellular water homeostasis and short-distance transport here. Long-distance transport of water is covered in Chap. 10.

6.2.1 The Water Potential

The thermodynamic state of water is described by the water potential (Ψ _w), which is, figuratively speaking, a measure of the energy required to remove water molecules from any water-containing system. The (chemical) potential of pure water under standard conditions of pressure and temperature is defined as zero and used as a reference. Any system that requires more energy to remove water from it has a negative water potential. Commonly, the water potential of a system is expressed in the dimension of pressure and not of energy: relating the chemical potential of water to the molar volume results in the dimension of “pressure”. Thus, Ψ_w of pure water under standard conditions is 0 MPa. Other details and the derivation of the definition of water potential are given in Chap. 10 (see also plant physiology textbooks).

The water potential of a solution, and correspondingly that of a cell, is influenced by three major components: concentration of solutes, pressure and gravity. When one is considering water potential beyond the cellular level or in cells and other structures in an at least partially dehydrated state, the matrix potential (Ψ _m) is often included as a fourth component. It describes the reduction in the free energy of water when it is adsorbed in a thin layer to surfaces of cell walls, soil particles or other structures. In the analysis of cellular processes it becomes apparent only after the cell has been desiccated to such an extent that only the water bound to cellular and subcellular structures remains. Removal of that portion of cellular water requires either extremely low (negative) water potentials or extremely high pressures. Because of very small values of the matrix potential in hydrated cells, it can be neglected for the discussion of cellular water relations, with the exception of freezing dehydration (Chap. 4, Box 4.1).

The concentration of solutes translates into the osmotic potential (Ψ _s). Jacobus H. van ʼt Hoff (who received the Nobel Prize for Chemistry in 1901) was the first to quantitatively describe the linear relationship between solute concentrations and the osmotic pressure exerted by water flowing through a semipermeable membrane separating two aqueous solutions (semipermeable = permeable for water molecules but not for the ions and molecules dissolved in water). Osmotic pressure is positive, while osmotic potential has a negative sign. It describes the negative effect of dissolved ions or molecules on the water potential. In other words, solutes reduce the probability of a water molecule moving across a semipermeable membrane into another compartment. The net flow of water between two solutions is directed towards the solution with the higher solute concentration.

The pressure potential (Ψ _p) refers to the hydrostatic pressure of a solution. A positive value raises the water potential—that is, the tendency of a water molecule to move from one place (e.g. a cell) to another. A positive hydrostatic pressure in a plant cell is equivalent to the turgor pressure. It is generated by the osmotically driven influx of water into a cell and the limited extensibility of the cell wall. Some cells in a plant can have negative hydrostatic pressure (tension). This applies, for instance, to xylem vessels. Both positive and negative pressures are made possible by the rigidity of cell walls.

The influence of gravity, which forces water molecules downward towards the Earth’s centre, is expressed with the term Ψ _g. When analysing plant–water relations the water potential is mathematically often described as:

${\varPsi}_{\mathrm{w}}={\varPsi}_{\mathrm{s}}+{\varPsi}_{\mathrm{p}}+{\varPsi}_{\mathrm{m}}+{\varPsi}_{\mathrm{g}}$

An alternative formula to describe the water potential is explained in Chap. 10.

Ψ_g needs to be included when vertical movement of water over long distances (such as in tall trees) is analysed. Near the base of a plant, Ψ_g is negligible, so a discussion of plant–water relations at the cellular level can focus on Ψ_s and Ψ_p. In order to understand the movement of water into a plant, within a plant and out of a plant, one has to determine ΔΨ_w (i.e. the water potential difference) between relevant places—for example, the soil and root cells, root cells and mesophyll cells, or mesophyll cells and the atmosphere.

6.2.2 Facilitation of Intercellular and Intracellular Water Flow: Aquaporins

Water movement in a plant is always passive. It follows gradients in potential. A water potential difference between two systems (e.g. two cells) can be compared to a voltage in an electrical circuit. It causes the flux of water in the direction of the system with the more negative potential, provided that a water conductive pathway exists between the systems. In accordance with the analogy to an electrical circuit, the pathway for water corresponds to a resistance. While long-distance movement of water occurs mostly through vascular cells (Chap. 10), three pathways for the flux of water through a tissue exist. Let us, for instance, consider the radial movement of water through the root towards the vasculature. The cells through which this radial movement occurs represent an important resistance for water flow (Steudle and Peterson 1998). It is therefore physiologically very important under conditions of transpiration when water has to be replenished from the soil solution. The flux of water may occur in the apoplast—that is, through and along the cell walls (the apoplastic path)—or from cell to cell via plasmodesmata (the symplastic path). The third path is transcellular and requires membrane passages (Fig. 6.3). The different pathways and respective forces driving the water flux are integrated into the “composite water transport model” (Steudle 2001). Apoplastic water flux is mostly due to hydrostatic forces (pressure or tension). In contrast, the symplastic and transcellular paths are additionally driven by osmotic pressure, as osmotic gradients can establish across the cellular membranes. The relative contributions of the three pathways vary widely depending on the species, developmental stage or environmental conditions. For instance, in a transpiring plant, hydrostatic forces dominate, while in the absence of transpiration, osmotic pressures are more important (Javot and Maurel 2002).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig3_HTML.png — Fig. 6.3
Three pathways of water flow through living plant tissues: apoplastic, symplastic and transcellular. (Modified from Javot and Maurel (2002))

Movement of water through cellular membranes is largely controlled by water channels—the aquaporins (Chaumont and Tyerman 2014). Their crucial role for water flow within tissues has been clearly determined. A widely used method to differentiate between the movement of water molecules directly through the membranes (which is the essence of the semipermeability of biological membranes) and aquaporin-dependent water movement is to compare conductivities in the absence and presence of mercury ions (Hg²⁺). Hg²⁺ oxidises crucial cysteine residues in aquaporins and thereby inactivates them. The effect can be reversed by thiol reagents (Fig. 6.4). Hg²⁺ exposure strongly reduces the water flow through roots. Thus, a large (yet variable) fraction of root water permeability is attributable to aquaporins.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig4_HTML.jpg — Fig. 6.4
Effects of mercury (Hg²⁺) on pressure-induced water transport in excised tomato roots. Sap flux through roots of de-topped tomato plants was continuously monitored. The plants were held under constant pressure. HgCl₂ (0.5 mM) and the reductant β-mercaptoethanol (ME; 60 mM) were applied at the times indicated. Hg²⁺ blocks aquaporins via the interaction with conserved cysteines; β-mercaptoethanol reverses this effect. The sap fluxes were recorded in an HgCl₂-treated root system (*closed circles*) and a corresponding untreated control root system (*open circles*). HgCl₂ reduced the intensity of sap flux in a partially reversible manner by about 70%. This reduction was assumed to reveal the contribution of aquaporins to root conductivity in this experiment. (From Javot and Maurel (2002), after Maggio and Joly (1995))

Aquaporins represent an ancient family of small membrane proteins. They consist of six membrane-spanning helices connected by five loops (Fig. 6.6). The N– and C–termini are cytosolic. Their molecular mass lies between 21 and 34 kDa. Shortly after their unexpected discovery in the early 1990s in animal cells (for which Peter Agre received the 2003 Nobel Prize in Chemistry; Agre 2004), they were also identified in plants and microorganisms. Aquaporins transport water molecules and also a range of other substrates such as CO₂, boron and silicon (Maurel et al. 2015). In accordance with the importance of controlled water movement in all types of cells at every developmental stage, plants possess a large number of isoforms (e.g. 35 in the eudicot Arabidopsis thaliana and 33 in the monocot rice) with distinct expression patterns and subcellular localisations (Maurel et al. 2008). Most abundant are aquaporins in the plasma membrane (PIPs (plasma membrane intrinsic proteins)) and in the tonoplast (TIPs (tonoplast intrinsic proteins))—that is, the two membranes that most of the transcellular water movement goes through. However, they are also found in all other membranes of the endomembrane system.

The principal ways of controlling aquaporin-dependent water flow are modulation of aquaporin abundance and permeability (= gating). Across a plant organ, aquaporin expression levels are not equal in all cell types. Instead, it can be observed that certain cells and cell types show higher abundance of aquaporins. These are cells of particular importance for controlling the flow of water. They function as gatekeepers (Chaumont and Tyerman 2014) and include the stomata (Sect. 6.3.3), bundle sheath and xylem parenchyma cells in the leaves. In roots, gatekeeper cells are located near the endodermis and the exodermis. The apoplastic pathway through the root is blocked by these cell layers. Suberised and lignified cell walls (referred to as the Casparian stripin the case of the endodermis) prevent movement of water molecules along the cell wall. Entry into the symplastic pathway requires a membrane passage that is facilitated by aquaporins.

Especially given the existence of gatekeeper cells, it is evident that the passive flow of water through a plant can be regulated by the abundance and activity of aquaporins. Coming back to the electrical circuit analogy, aquaporins represent variable resistances. The voltage (the water potential gradient) cannot be influenced, but the current (the flow of water) can. One way of modulating the “resistance” is transcriptional control over aquaporin expression levels. For instance, diurnal and circadian rhythms in the conductivity of roots and leaves can be well explained by corresponding changes in the transcription of major PIP genes (Maurel et al. 2008) (Fig. 6.5). Such rhythmicity couples tissue water conductivity to stomatal functions. During the light period when stomata are open, the tissue conductivity is increased (the resistance is lowered) through higher expression levels of aquaporins, thereby preventing the emergence of extreme xylem tension due to transpiration. Various environmental stresses, including drought, affect aquaporin expression, often in complex ways. In addition to transcriptional control, several mechanisms for regulating aquaporin activity are known. Aquaporin residence time in the plasma membrane is influenced by constitutive recycling; that is, aquaporin molecules are trafficked not only in vesicles from the endoplasmic reticulum and Golgi to the plasma membrane but also in the reverse direction. In this way a cell can finely adjust the number of active aquaporins.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig5_HTML.png — Fig. 6.5
Diurnal rhythm of *Lotus japonicus* root conductivity and aquaporin expression. a A peak in root water conductivity near the middle of the light period, when stomata are open and transpiration is high, coincides with higher levels of aquaporin transcripts. b Northern blot showing changes in the abundance of a major plasma membrane aquaporin transcript, detected with a probe derived from the *Arabidopsis thaliana* homologue. This illustrates the coordination between transpiration and water conductivity of tissues, which is achieved by transcriptional regulation of aquaporins. (From Javot and Maurel (2002), adapted from Henzler et al. (1999))

Furthermore, the stability of aquaporins is modified by phosphorylation and ubiquitination. Gating of the water channels is modulated through heteromerisation, Ca²⁺ levels, pressure or pH. All of these processes can be influenced by environmental cues (Fig. 6.6) (Chaumont and Tyerman 2014). In summary, the multitude of aquaporin isoforms in a plant and the many ways of regulating them provide the means to acclimate to fluctuations in water availability and demand by adjusting resistance for water flow across tissues as well as into and out of cells and cellular compartments.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig6_HTML.png — Fig. 6.6
Regulation of plasma membrane aquaporin (plasma membrane intrinsic proteins (PIPs)) abundance and activity within the cell. *PIP* genes are transcribed, their messenger RNA (mRNA) translated in the rough endoplasmic reticulum (ER) and the proteins targeted at the plasma membrane (PM). PIPs of different classes (depicted in *yellow* and *green*) can form homo- or hetero-oligomers. PIP oligomers transit through the Golgi apparatus via the trans-Golgi network (TGN) and are then routed to the plasma membrane in secretory vesicles. Transcription and trafficking are modulated depending on the developmental stage and various environmental cues. Insertion of PIPs into the plasma membrane is mediated by the syntaxin SYP121. Internalisation of plasma membrane–localised PIPs occurs as a result of constitutive recycling. Once internalised in vesicles, PIPs are delivered to the TGN before being routed back to the plasma membrane or directed into lytic vacuoles for degradation. Under stress conditions, PIPs can be dephosphorylated and internalised, or ubiquitinated and then degraded in the proteasome. The water channel activity or gating of PIPs is regulated by different mechanisms (heteromerisation, phosphorylation, interaction with SYP121, protonation, the pressure gradient and the Ca²⁺ concentration). In the *upper right corner* the topological structure of an aquaporin monomer is shown (Murata et al. 2000), which consists of six membrane-spanning α-helices (1–6) connected by five loops (A–E) and N– and C–termini facing the cytosol. The loops B and E, together with the membrane-spanning helices, create a pore with high specificity. The transcription, translation, trafficking and gating of PIPs are regulated by environmental and developmental factors involving signalling molecules, phytohormones and the circadian clock. (Chaumont and Tyerman 2014)

6.3 Drought Stress Responses: Avoidance and Tolerance

Under conditions of drought, plants lose water to the atmosphere. When water uptake cannot keep pace with water loss, transpiration is fed mainly from the vacuoles. Usually the water permeability of the tonoplast is considerably higher than that of the plasma membrane. This allows fast equilibration of the intracellular water potentials upon changes in the cell’s water status. Water flows via the plasma membrane into the apoplast, from where it evaporates into the intercellular spaces. Because of the decrease in volume caused by water loss, the osmotic pressure in the protoplast is increased; that is, the osmotic potential becomes more negative. At the same time, the hydrostatic pressure decreases. When the so-called turgor loss point is reached, the cell wall is completely relaxed and the pressure (Ψ_p) is zero. Consequently, the water potential of the cell is then equal to its osmotic potential (Ψ_s). In this state a plant shows substantial wilting. With further loss of water, wilting increases, as the cell walls not only are relaxed but also, in responding to the intracellular suction, bend inwards. This will finally result in cytorrhysis—that is, the complete collapse of the cell—provided that the cell wall rigidity is low enough to allow folding. As described for the case of freezing dehydration, this happens because the water-imbibed cell wall (Sect. 4.2.6 in Chap. 4) does not allow any air to penetrate. Suction develops and, correspondingly, a negative water potential (−1 to −2 MPa) of the cell arises.

Besides the escape of water-limited conditions through the timing of, for instance, germination or flowering (Fig. 6.7) (Chap. 2), principally two different strategies to cope with water scarcity can be distinguished: avoidance and tolerance. It is important to note, however, that these categories merely represent distinct areas within a continuum of responses. They can even both be displayed by the same plant, depending on the severity of the water limitation (Fig. 6.7). Avoidance refers to a balancing of water uptake and water loss that maintains the water status. This can be achieved by restricting water loss, by increasing the water supply or by water storage. It has been argued that avoidance is an idealised concept. In reality, avoidance mechanisms achieve later onset of drought stress (Lawlor 2013). Tolerance mechanisms help a plant endure a moderate lowering of the water potential. They protect cells against damage potentially arising from water loss—for instance, by compatible solute accumulation (Sect. 6.3.1). Key cellular functions are maintained, enabling resumption of growth after resupply of water.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig7_HTML.png — Fig. 6.7
Drought adaptation strategies ranging from escape to tolerance of severe dehydration. (Modified from Verslues and Juenger (2011))

An extreme form of drought tolerance is shown by poikilohydric plants, which withstand near complete tissue dehydration (desiccation). Lichens and certain mosses, on the one hand, and resurrection plants such as Craterostigma plantagineum and Xerophyta humilis, on the other hand, have evolved specific adaptations that allow them to enter a dormant or quiescent state under dry conditions. These adaptations are substantially different from those that allow homoiohydric plants to continue their physiological activity during less severe water limitation. Owing to the accumulation of protective proteins (Sect. 6.3.2) and osmolytes, cells of poikilohydric plants survive a very low water status and are able to resume metabolic activity following rehydration. This enables poikilohydric plants to inhabit extremely dry environments such as the deserts in Southern Africa, where a diversity of resurrection plants is found.

Finally, another useful categorisation of strategies similar to the avoidance–tolerance distinction is the differentiation between isohydric and anisohydric plant species. It refers to two different types of stomatal regulation (Sect. 6.3.3) in response to soil drying. Isohydric plants such as maize or poplar maintain the water potential of their cells at relatively constant values (at around −2 MPa) by reducing stomatal conductance early upon the onset of water shortage. In contrast, anisohydric species (e.g. sunflower) allow a decline in cellular water potential upon a drought-induced drop in soil water potential. The two strategies are associated with different physiological risks. Rapid stomatal closure can lead to early onset of a negative carbon balance because of the reduced CO₂ assimilation. On the other hand, the decrease in water potential can result in hydraulic failure. When water loss through transpiration is substantially greater than the uptake of water by roots, high xylem water tension can develop, which eventually leads to cavitation of xylem vessels and conductivity loss (McDowell et al. 2008) (Chap. 10). Overall, anisohydric species tend to be more common in drought-prone habitats than isohydric species.

6.3.1 Control of the Osmotic Potential

When the water potential outside a cell is more negative than the water potential of the cell itself, a net flux of water out of the cell will occur. Thus, in order for the plant to take up water from the soil solution, the water potential of its root cells has to be more negative than that of the surrounding soil; that is, the cells have to establish a water potential difference. Under conditions of drought the soil water potential decreases substantially because of the adhesion of the remaining water to soil particles (a strongly negative matrix potential (Ψ_m) develops). This poses the challenge to maintain a water potential gradient. It is important to note that several other environmental conditions cause a very similar problem. The water potential of wet soil can become too negative when high concentrations of salt are present (Chap. 7). Likewise, freezing of extracellular water causes an extreme drop in the water potential (Chap. 4).

Plant cells can lower their water potential by osmotic adjustment. Increasing the concentration of solutes makes the osmotic potential (Ψ _s), and thereby the water potential of the cell, more negative. Osmolality increases which are achieved in this way exceed the solute-concentrating effect of partial dehydration. The process of preventing cellular water loss by achieving a more negative water potential than that of the surrounding solution is an acclimative response and is referred to as osmoprotection. It is known from all kinds of organisms (Yancey 2005). The solutes accumulating for osmotic adjustment are called “compatible solutes” or “osmolytes”. They are mostly organic low molecular weight compounds. Accumulation of ions would principally have the same effect on the osmotic potential of a cell. However, elevated concentrations of ions may affect the hydration shell of proteins and thereby inactivate them. In contrast, many organic molecules are compatible with cell functioning because they are either uncharged or zwitterionic at physiological pH. They do not enter the hydration shell of proteins and, owing to their hydrophilicity, they can become a constituent of the structured portion of the water film on the membrane surface. Compatible solutes accumulate predominantly in the cytosol and in metabolically active organelles such as the chloroplasts to maintain an osmotic balance with the vacuoles where ions can be stored and lower the osmotic potential without disturbing metabolism.

The osmotic potential of a cell is a function of the concentrations of a vast number of dissolved molecules and ions. Only a few of them accumulate massively (up to about 10% of the dry weight) for osmotic adjustment and therefore function as osmolytes. They are chemically diverse and predominantly belong to the soluble sugars (e.g. glucose, sucrose), sugar alcohols (e.g. mannitol, sorbitol), oligosaccharides (raffinose, stachyose), amino acids (e.g. proline), quaternary ammonium compounds (e.g. glycine betaine) or polyamines (e.g. putrescine, spermidine) (Table 6.2). Some of these metabolites accumulate in many different plant species of diverse phylogeny. Proline is a prominent example (Fig. 6.8). Other osmolytes are typical for particular plant families. For instance, β-alanine betaine as an osmolyte is largely confined to Plumbaginaceae; glycine betaine is common in Chenopodiaceae. Thus, different molecules have been recruited during evolution for the purpose of osmotic adjustment.

Table 6.2

Accumulation of molecules in organs of terrestrial plants under drought stress, and their physiological effects

Type of molecule	Examples	Function under drought
Ions	K⁺	Osmotic adjustment
Proteins	LEA/dehydrins	Membrane and protein protection
Proteins	SOD, catalase	ROS detoxification
Metabolites
Amino acids	Proline	Membrane and protein protection
Sugars	Raffinose	ROS scavenging
Polyols	Mannitol (acyclic)	Osmotic adjustment, ROS scavenging
Polyols	Pinitol (cyclic)	Osmotic adjustment, ROS scavenging
Polyamines	Spermine	ROS scavenging, membrane protection
Polyamines	Spermidine	ROS scavenging, membrane protection
Quaternary amines	Glycine betaine	Membrane and protein protection
Quaternary amines	β-Alanine betaine	Osmotic adjustment under hypoxia
Tertiary sulphonium compounds	Dimethyl sulphonopropionate	ROS scavenging
Pigments	Carotenoids	Protection against overexcitation, photoinhibition
Pigments	Anthocyanins	Protection against overexcitation, photoinhibition

LEA late embryogenesis abundant, ROS reactive oxygen species, SOD superoxide dismutase

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig8_HTML.png — Fig. 6.8
The compatible solute proline. Time course of accumulation and decrease in proline (Pro) concentration and the messenger RNAs for D1-pyrroline-5-carboxylate synthase (P5CS) and for proline dehydrogenase (proline oxidase (ProDH)) in *Arabidopsis thaliana* during the development of drought a and 10 h after rehydration b. (After Yoshiba et al. (1997))

The protective function of osmolytes goes beyond the osmotic effect (Table 6.2). Unlike the lowering of the osmotic potential, however, other activities cannot generally be ascribed to all osmolytes. Instead, they are more specific to particular compound classes (Yancey 2005). Some osmolytes—for example, mannitol and other sugar alcohols—are believed to act as antioxidants—that is, to scavenge reactive oxygen species (ROS) generated during drought or freezing. Others, such as proline and β-alanine betaine, have been implicated in redox balancing. In this case it is not a characteristic of the solute itself that explains this protective function; rather, the synthesis of proline and other solutes is reductive. The oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) regenerates NADP⁺ as an electron acceptor. This reduces the risk of electron transfer from photosystem I to O₂ and thereby lowers the probability of photoinhibition. Proline may also function as a molecular chaperone, stabilising proteins and other macromolecules. Protection of enzymes involved in detoxification of ROS would then offer yet another explanation for the documented antioxidant effect of proline (Szabados and Savouré 2010). Similar protective effects on enzymes that are important either for stress tolerance or for metabolic activity under stress have been described for glycine betaine too.

The accumulation of osmolytes under conditions of drought and other stresses such as freezing (Chap. 4) and osmotic stress (Chap. 7) has been documented in countless studies for a wide range of plant species. Nevertheless, direct unequivocal evidence demonstrating the importance of these acclimative responses—for example, through a loss-of-function mutation and concomitant stress hypersensitivity—has not been possible to obtain. This is different from the modulation of stomatal conductance. Inability to close stomata under drought stress dramatically accelerates the wilting of plants (Sect. 6.3.3). The importance of compatible solute accumulation has initially been mostly inferred from similar responses in microorganisms such as Saccharomyces cerevisiae, Escherichia coli or salt-tolerant cyanobacteria and the demonstrated contribution of compatible solutes to survival in these biological systems. Experimental support in plants has predominantly come from studies with transgenic plants engineered to overproduce particular compatible solutes. Very often such plants showed moderate gains in stress tolerance. Analyses of extremophile species with extraordinary levels of abiotic stress tolerance have provided some additional indirect evidence. The salt-tolerant Brassicaceae Eutrema salsugineum (formerly Thellungiella halophila), for instance, has higher proline levels already in the non-stressed state and accumulates proline more strongly under stress than other related Brassicaceae species (Chap. 7). Similarly, levels of pinitol are higher in some drought- and salt-tolerant species such as Mesembryanthemum crystallinum. It cannot be generalised, however, that particularly stress tolerant plant species always display stronger accumulation of compatible solutes.

The strong increase in osmolyte accumulation requires many changes in the transcriptome, proteome and metabolome of plants exposed to water deficit stress. Accumulation can arise in different ways. Monosaccharide concentrations can be adjusted through the breakdown and synthesis of starch and other polysaccharides. For sugar alcohols such as mannitol the utilisation of the hexose phosphate pool is diverted away from sucrose synthesis by the induction of enzymes that use fructose-6-phosphate for sugar alcohol synthesis. Proline concentrations are a function of synthesis and degradation rates (Fig. 6.8). Their regulation has been studied intensively. As mentioned earlier, proline is a multitasking molecule with many physiological functions besides its role as one of the 20 amino acids found in proteins. Correspondingly, several different environmental cues influence the rates of synthesis from glutamate and the degradation back to glutamate. Some of them are shown in Fig. 6.9 (the alternative biosynthetic pathway from ornithine is not depicted). The major hormone eliciting drought responses is abscisic acid (ABA) (Sect. 6.5). When it accumulates in cells, pyrroline-5-carboxylate synthetase (P5CS)—the enzyme catalysing the first step in the synthesis from glutamate to proline—becomes transcriptionally up-regulated. In A. thaliana the drought responsive gene is P5CS1. The second isoform, P5CS2, is not activated even though it is highly similar to P5CS1. It has housekeeping functions; that is, it is responsible for the proline synthesis that is needed in the absence of stress. Under salt stress, P5CS1 is activated via a different signal transduction pathway (Szabados and Savouré 2010). Catabolism is repressed under drought stress conditions, while it is stimulated upon stress relief—for instance, by rehydration. Control is exerted on two enzymes, proline dehydrogenase (PDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig9_HTML.jpg — Fig. 6.9
Proline synthesis and degradation in *Arabidopsis thaliana* are responsive to several environmental cues. The cues depicted here are water status and salt stress. The influences of other factors such as light, the photoperiod and pathogen attack, as well as the complex cellular compartmentation of proline metabolism, are not shown. Synthesis of proline (*green lines*) from glutamate is stimulated by the transcriptional up-regulation of one particular pyrroline-5-carboxylate synthetase isoform (*P5CS1*) via different signal transduction cascades depending on the type of water deficit stress (drought versus high salt concentrations in the soil). Activation under drought stress is mediated by abscisic acid (ABA) and reactive oxygen species (ROS) as signalling molecules. Pyrroline-5-carboxylate reductase (P5CR) is up-regulated under drought stress. The end product proline feedback-inhibits P5CS1. Proline degradation (*red lines*) to glutamate by proline dehydrogenase (PDH) and pyrroline-5-carboxylate dehydrogenase (P5CDH) is inhibited under drought stress and stimulated upon rehydration. The *P5CDH* transcript is targeted by a natural small interfering RNA (siRNA) upon drought activation of *SRO5* transcription. Regulatory proteins involved in signal transduction cascades are shown in *hexagons*: transcription factors of the bZIP and MYB class, calmodulin (CaM), the phosphatase ABI1 and phospholipase C (PLC). (Modified from Szabados and Savouré (2010))

6.3.2 Protective Proteins

The function of proteins accumulating under conditions of drought have been mostly studied in the context of desiccation—that is, a lowering of the relative water content of a tissue or organ to 10% or less. Desiccation tolerance represents an adaptation to extreme environmental conditions and requires specific mechanisms displayed by only a small number of plant species (around 300 within the angiosperms). This makes it different from the acclimative control of osmotic potential or the stomatal aperture (Sect. 6.3.3), which essentially every plant is capable of. It involves a state of dormancy of the whole plant, which is reminiscent of seeds. In fact, it has been proposed that in resurrection plants the developmental programmes underlying seed maturation have been recruited for the desiccation tolerance of the whole plant (Farrant and Moore 2011).

During the early phase of dehydration, osmotic adjustment occurs through the accumulation of sucrose and other osmolytes. Progressive water loss concentrates the cellular content and causes mechanical as well as metabolic stress. The risk of protein denaturation and unwanted biochemical reactions increases. Osmolytes replace water molecules and can eventually accumulate to levels such that vitrification occurs; that is, the sucrose solution adopts a glassy, almost solid state (Fig. 6.10). Proteins that have the capacity to protect cellular structures strongly accumulate. They are called late embryogenesis abundant (LEA) proteins. The name indicates that these proteins were first described as accumulating during the later stages of seed maturation, when desiccation tolerance is acquired. However, they are found not only in plants but also in bacteria, archaea, fungi and certain invertebrates (e.g. nematodes, arthropods).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig10_HTML.png — Fig. 6.10
Changes in the proportions of soluble carbohydrates, corresponding to the water relations of the resurrection plant *Craterostigma plantagineum*. In the fully hydrated state, leaves mainly contain the C8 sugar octulose which, upon desiccation, is almost quantitatively converted to sucrose, a compatible solute. This reaction is reversible. (After Bartels et al. (1993))

Within the plant kingdom, LEA proteins are not specific to resurrection plants. They accumulate in the seeds of most other plants during ripening. Also, their expression is induced under conditions of water limitation such as drought, freezing and high salt concentrations in the vegetative tissue of many non-desiccation-tolerant plant species. Some of the COR (COld-Regulated) genes up-regulated during cold acclimation (Chap. 4) encode LEA proteins. Genome-wide analyses in model species have shown that different subsets of LEA genes are expressed during seed maturation and abiotic stress with very little overlap. Resurrection plants (Fig. 6.11) are special with respect to LEA proteins only in that they often show much stronger accumulation of these proteins. Also, in some species, these proteins are constitutively expressed at high levels even in the absence of water deficit. The latter is interpreted as a way of priming these plants for dehydration upon the arrival of severe drought events.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig11_HTML.png — Fig. 6.11
The resurrection plant *Craterostigma plantagineum*, which recovers fully from drying out: a plant in the hydrated state (*left*), in the dry state (*centre*) and after rehydration (*right*). (Photos courtesy of Dorothea Bartels, University of Bonn, Germany)

LEA proteins are highly hydrophilic. On the basis of their amino acid composition (for instance, a high content of glycine and other small amino acids) and supported by biochemical investigations of a few examples, most of them are assumed to be intrinsically disordered proteins—that is, lacking a tertiary structure. Large numbers of genes encoding LEA proteins are found in plant genomes (>50 in A. thaliana, for example). LEA proteins are divided into several classes according to sequence similarities. The annotation and categorisation of LEA proteins are not consistent throughout the literature. They largely overlap with another group of proteins named “hydrophilins”, which are defined as glycine rich and hydrophilic. Among the different classes of LEA proteins there are some that carry alternative names such as “dehydrins”.

The increase in LEA protein abundance under water deficit conditions has been documented for a large number of plant species, organs, tissues and cell types. LEA proteins have thus been firmly associated with abiotic stress tolerance. Still, the functional understanding of LEA proteins is limited. How exactly their accumulation promotes cell survival under stress is unknown (Wise and Tunnacliffe 2004). Enzymatic activities have never been described. Many LEA proteins become structured—that is, partially folded—upon dehydration and may exert their protective effects in this state. On the other hand, the accumulation in water-limited vegetative tissues not undergoing desiccation suggests functions of LEA proteins also in the hydrated state. Several hypotheses exist as to the actual physiological and biochemical activities, including the stabilisation of proteins and membranes, anti-oxidative activities or a function as space-filling molecules in cells with low water content to prevent collapse.

In vitro it has been shown for several enzymes that the presence of LEA proteins preserves activity, probably because the hydrophilicity of LEA proteins prevents the formation of protein aggregates. This is sometimes referred to as a “molecular shield” mechanism and is different from the chaperone activity of heat shock proteins (Chap. 4), as LEA proteins cannot protect proteins from heat denaturation. According to this hypothesis the unstructured LEA proteins would sterically hinder the interaction between partially denatured proteins. The anti-aggregate function is supported by in vivo data obtained for cells co-expressing LEA proteins and aggregation-prone target proteins. Evidence for an interaction of LEA proteins with membranes exists as well. Binding may occur concomitantly with the formation of α-helices upon drying or alternatively in a hydrated state. Certain dehydrins have been found to electrostatically interact with membrane lipid head groups in solution. Direct evidence for stress protection conferred by LEA proteins is rare. Numerous studies with plants ectopically expressing LEA proteins have reported comparatively modest gains in stress tolerance.

Besides LEA proteins, other protective proteins accumulate in resurrection plants and generally in plant cells affected by water loss. The most prominent ones are small heat shock proteins (sHSPs). They show true chaperone characteristics in addition to a general stabilising effect on cellular macromolecules and membranes. A third group of protective proteins comprises ROS scavenging enzymes such as aldehyde dehydrogenases and peroxiredoxins (Chap. 2) to counteract the production of ROS in cells affected by water loss.

6.3.3 Regulation of the Stomatal Aperture

Leaf surfaces sealed by a cuticle and wax deposition represent a key innovation for the evolution of land plants. Without an effective barrier against water loss the maintenance of a relatively constant internal water status (homoiohydry) would not be possible in most terrestrial habitats. While there is considerable variation in the effectiveness of the sealing—for example, between plants inhabiting wetlands and xerophytes thriving in arid habitats—the cuticular water conductance rarely accounts for more than a small fraction (10% or less—much less in the case of xerophytes with a thick cuticle and massive wax deposition) of total evaporation. Most of the gas exchange and, with that, most of the water loss are due to stomatal conductance. It has been estimated that about 60% of all terrestrial rainfall globally is returned to the atmosphere through stomata. In water-limited ecosystems this proportion can be even higher (Katul et al. 2012) (Chap. 9). Because of the large difference in water potential between leaves and air (with the air having very negative values), the control of the stomatal aperture is the most important response to conditions of low water availability—for example, a more negative soil water potential. Regulation of stomatal conductance therefore plays a key role in a plant’s response to water deficit and drought stress tolerance. Plants that are unable to close their stomata die quickly when water is withheld.

Several internal and environmental cues are integrated by guard cells (which form the stomatal pore) in order to optimally adjust the stomatal aperture for any given physiological situation. Stomatal opening is in most plants (with the exception of CAM plants; Sect. 6.6) triggered by light. Stomatal closure under conditions of water limitation is elicited by the phytohormone abscisic acid (ABA) (Fig. 6.12) (for ABA signal transduction, Sect. 6.5). Other factors influencing the stomatal aperture are temperature (with lower temperatures favouring opening) and internal CO₂ (with higher CO₂ partial pressure favouring closing). Recognition of microorganisms via microbe-associated molecular patterns (MAMPs; Chap. 8) leads to stomatal closure because stomata represent important entry sites for pathogens.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig12_HTML.jpg — Fig. 6.12
Light-induced and abscisic acid (ABA)–induced stomatal movement in the abaxial epidermis of intact tobacco leaves. a At t = 0 the leaves were illuminated with white light. The images shown in c correspond to the open symbols on the graph in a. b ABA (10 μM) was applied to the leaf cuticle. The images shown in d correspond to the closed symbols in b. Note the different response kinetics. (Kollist et al. 2014)

The stomatal aperture is nearly linearly correlated with the guard cell turgor pressure. Guard cells are built in such a way that higher turgor pressure leads to a bending of the cells and thereby an opening of the pore between them. The turgor pressure is a function of the osmotic potential of the cells. A more negative value relative to the apoplast results in water influx, and vice versa. Thus, the concentration of solutes in guard cells determines stomatal conductance. Stomatal movement is driven by the transport, as well as the synthesis and degradation, of solutes. Most important are K⁺ ions and their counter ions Cl⁻ and malate²⁻. Changes in K⁺ and Cl⁻ concentrations are brought about by ion channel–mediated exchange between guard cells and the surrounding apoplast, as well as between guard cell vacuoles and the cytosol. Malate, in contrast, is either synthesised from starch or degraded via mitochondrial respiration either in guard cells or in neighbouring epidermal cells. Because the majority of solutes are stored in the vacuoles, the control of stomatal movement depends on the modulation of transport activities in both the plasma membrane and the tonoplast.

The principal classes of transporters are described in Chap. 7. Negative membrane potentials across the plasma membrane and the tonoplast are generated by different types of proton pumps: P-type H⁺-ATPases in the plasma membrane and V-type ATPases and pyrophosphatases in the tonoplast (Fig. 6.13). K⁺ influx and efflux is mediated by K⁺ channels, whose activity is dependent on the plasma membrane potential. Inward-rectifying channels (i.e. channels allowing the passage of ions more easily into the cell) open at membrane potentials more negative than the resting potential for K⁺ (i.e. upon hyperpolarisation) and mediate K⁺ influx. Outward-rectifying K ⁺ channels (i.e. channels allowing the passage of ions more easily out of the cell) open at membrane potentials more positive than the resting potential for K⁺ (i.e. upon depolarisation) and mediate K⁺ efflux. K⁺ uptake into the vacuole occurs against a concentration gradient and the electrical potential difference. Thus, it is assumed to require K⁺/H⁺ symport. Efflux from the vacuole is channel mediated.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig13_HTML.png — Fig. 6.13
Plasma membrane–localised and tonoplast-localised channels and transporters involved in opening and closing of stomata. Proton pumping activities of P-type H⁺-ATPases, V-type H⁺-ATPases and pyrophosphatases result in an acidic pH of the vacuole and the apoplast. Opening of stomata is triggered by a stimulation of proton pumping that renders the potential of the plasma membrane more negative (approximately −120 mV), which activates inward-rectifying K⁺ channels (K_in channels). Influx of anions is dependent on H⁺-driven symporters such as the NRTs in the case of NO₃ ⁻. The K⁺ ions taken up into the cytosol are transported into the vacuole by NHX transporters—antiporters driven by H⁺ flux. Anions and malate are stored in the vacuole, following passage through anion channels (ALMT) or transporters (CLC). Stomatal closure is initiated by the activation of anion channels. The efflux of anions out of the cell depolarises the plasma membrane to about −60 mV. Outward-rectifying K⁺ channels open and K⁺ efflux occurs. (Modified from Kollist et al. (2014))

Because of the voltage dependence of K⁺ channels in the plasma membrane, K⁺ movement into and out of guard cells is controlled by the plasma membrane potential. Hyperpolarisation is achieved by increases in proton pumping activity. Light-triggered stomatal opening is dependent on blue light receptors (phototropins), which further activate H⁺-ATPases, leading to hyperpolarisation, K⁺ influx and, finally, osmotically driven water uptake. There is also evidence for an opening in response to photosynthetically active radiation, which is mechanistically poorly understood. The actual signal sensed by the guard cells could be the lowering of the CO₂ concentration in the sub-stomatal cavity owing to active photosynthesis. In this way, CO₂ demand would be coupled to the stomatal aperture.

ABA-triggered stomatal closure is mediated by an inactivation of proton pumps and a concomitant activation of Cl⁻ channels (such as SLAC1) in the plasma membrane. The resulting depolarisation opens the outward-rectifying K⁺ channels and thereby causes K⁺ efflux and water loss into the apoplast (Fig. 6.14). ABA signal transduction is discussed in Sect. 6.5. Other factors triggering stomatal closure include low humidity and elevated atmospheric CO₂ levels (Chap. 10).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig14_HTML.png — Fig. 6.14
Abscisic acid (ABA)–dependent guard cell ion channel regulation. ABA triggers stomatal closure (shown on the *left*) and inhibits stomatal opening mechanisms (shown on the *right*), which are activated, for instance, by blue light. *AHA1* ARABIDOPSIS H⁺ ATPASE 1, I _Ca inward Ca²⁺ current, *OST2* OPEN STOMATA 2, *R-type* rapid-type, *SLAC1* SLOW ANION CHANNEL 1, *S-type* slow-type, SV slow vacuolar, *TPK1* TWO PORE K⁺ CHANNEL 1, VK vacuolar K⁺ selective. (Kim et al. 2010)

6.4 Acclimation of Growth

An integral part of a plant’s acclimation to water deficit caused by drought or high salt—and essentially an avoidance strategy—is the reduction of leaf area relative to biomass. In this way the loss of water via transpiration is reduced and the water status of the plant is improved. Indirectly this strategy also lowers the risk of overheating that accompanies the closing of stomata. Unfortunately for a plant, this water-saving strategy comes with the cost of potentially lower reproductive success because fewer resources can be accumulated during the vegetative stage to produce viable seeds. The other problem is the risk of being outgrown by faster-growing competitors. It therefore represents one of the fundamental challenges for plants to find the right balance between investment in stress tolerance on the one hand and growth on the other hand (Fig. 6.15) (Claeys and Inzé 2013) (Chap. 2). Accordingly, a wide natural variation exists between species, and even within species, with respect to the thresholds of water supply that trigger a strong reduction or even halt of leaf growth. Depending on the extent of the stress, different strategies can be successful. Continued growth can be beneficial in comparatively mild water limitation scenarios but detrimental when a plant is exposed to a longer drought.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig15_HTML.jpg — Fig. 6.15
The trade-off between stress tolerance and maintained growth. In response to water limitation, stress avoidance and tolerance mechanisms are activated to ensure survival in case the stress is prolonged or becomes more severe, resulting in growth limitation and a potential competitive disadvantage. On the other hand, acclimative mechanisms exist that allow continued growth in situation where the stress is less severe. (Modified from Claeys and Inzé (2013))

A second acclimation of growth under conditions of water scarcity is the stimulation of root growth in order to improve the water uptake capacity and to access additional water resources. This strategy is viable as long as there is soil water available. Under more severe drought, root growth also becomes inhibited. The plasticity of roots in response to drought goes beyond the size. The root system architecture—that is, the combination of the primary root length, lateral root formation, and root hair density and length, as well as root diameters—is highly flexible and can be adjusted in response to environmental fluctuations (Chaps. 2 and 7).

It is one of the major principles of plant stress tolerance that growth reduction under unfavourable conditions is not simply a consequence of disturbance or damage but, rather, an active modulation of resource utilisation (Chap. 2). The slowing of growth is a result of balanced hormonal control. Molecular understanding of underlying mechanisms has developed rapidly and is summarised in this chapter with regard to drought.

6.4.1 Inhibition of Shoot Growth

Shoot growth responds very rapidly to water deficit. Acclimative slowing of growth can be observed within 20–30 min. The response occurs even in the absence of any changes in the water potential of the elongating cells. Thus, it is not a mere consequence of turgor loss. Furthermore, growth halts much faster than photosynthesis under drought conditions. A shortage of reduced carbon can therefore be ruled out as the cause of the growth reduction as well. In fact, sugars often accumulate after the onset of stress, meaning that growth is uncoupled from the availability of carbon under these conditions, once more indicating controlled processes rather than simple supply-driven processes. An important inference from active growth modulation is that growing tissues and mature tissues—for example, young sink leaves and old source leaves—respond differentially to stress (Fig. 6.16). This principal difference is indicated by many observations but has not been systematically dissected yet at the molecular level (Claeys and Inzé 2013).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig16_HTML.png — Fig. 6.16
Processes involved in growth regulation upon drought. The responses differ between mature and growing leaves and, in the latter, between proliferating leaves (growth by cell division) and expanding leaves (growth by cell expansion). Soil drying is sensed in roots, activating a combination of hydraulic and chemical signals, which are transported through the xylem to the leaves (Fig. 6.18), where they initiate a number of tolerance mechanisms. The response of mature leaves can be described by the avoidance/tolerance concept. In growing leaves, stress leads to acute growth inhibition followed by growth acclimation, both mediated by DELLA signalling—that is, the inhibition of gibberellic acid signalling. Ethylene promotes DELLA stabilisation and growth inhibition. While hormonal signalling is common between expanding and proliferating leaves, effector genes are distinct. In proliferating leaves, inhibitors of the cell cycle might play a role in the acute response. In expanding leaves, cell wall tightening and changes in cell turgor lead to growth cessation, while osmotic adjustment and cell wall loosening are important for growth acclimation. (Modified from Skirycz and Inzé (2010))

Growth is determined by the cell division rate and cell expansion rate. Under water-limited conditions, both processes are actively modulated in shoots. Cell cycle activity, which determines the cell division rate, is triggered by cyclin-dependent kinases and associated cyclins (see molecular biology and biochemistry textbooks). The major pathways controlling cyclin-dependent kinases are all responsive to drought. For instance, repression of the anaphase-promoting complex/cyclosome (APC/C), a master negative regulator of cell cycle activity in eukaryotes, is down-regulated, leading to reduced cyclin-dependent kinase activity. The cell cycle control pathways themselves are controlled by DELLA proteins as central regulators of gibberellic acid signalling and growth (Chap. 2). The stress hormone ethylene has also been implicated in cell cycle arrest. A very early transcriptional response to osmotic stress specifically in growing leaves is the up-regulation of ethylene response factors that stimulate the catabolism of gibberellin (Claeys and Inzé 2013).

Cell expansion requires turgor pressure and sufficient extensibility of the cell wall. Aquaporin-facilitated water uptake can lead to cell enlargement, provided that the osmotic driving force for water uptake—that is, the intracellular concentration of solutes such as K⁺—is strong enough. The higher the rigidity of the cell (the lower the extensibility of the cell), the higher the pressure required to expand the cell. The dynamics of cell wall characteristics therefore play a central role in plant growth processes. Cell wall flexibility is modulated, on the one hand, by ROS-dependent cross-linking and cleavage reactions and, on the other hand, by enzymes modifying the structure of pectins or influencing the interaction between cellulose microfibrils and hemicelluloses (for cell wall structure, see plant biochemistry and plant physiology textbooks). Such enzymes include pectin methylesterases, xyloglucan endotransglucosylases/hydrolases and expansins. Their expression patterns change substantially under conditions of drought (Tenhaken 2015). The precise and high-resolution description of cell wall dynamics and growth processes at the cell, tissue and organ levels is a complex systems biology problem, which is very actively studied and far from being solved. However, three aspects can be singled out to help illustrate the shoot growth responses under conditions of water scarcity. First, osmotic adjustment can maintain the turgor pressure to enable continued growth under stress and may thus have different significance for cells of growing leaves than for those of mature leaves. In the latter, the main purpose of osmotic adjustment is avoidance of water loss. Second, cell expansion with reduced turgor pressure can be achieved by increasing the flexibility of the cell wall. Third, growth can be effectively stopped by a stiffening of the cell wall. Shoot growth under drought often goes through two different phases. During the acute phase, growth ceases as a consequence of cell wall stiffening. In a second phase of growth acclimation the cell wall becomes more flexible again to support new expansion growth (Skirycz and Inzé 2010). Depending on the severity of stress and on the genetic variation selected for in habitats varying in the extent of water limitation, different processes can be dominant to establish the right balance for the fundamental trade-off between growth and stress tolerance. For instance, a detailed study of A. thaliana responses showed that under mild drought stress the expression of expansins was up-regulated (that is, cell walls became more extensible and growth was maintained) whereas under more severe drought, expansins were down-regulated (Harb et al. 2010) (that is, cell walls were more rigid and growth was halted).

Plants not only reduce the growth of evaporative surfaces when they are water limited; they also lower the density of stomata per epidermal cell and per unit of leaf area to further reduce water loss. In developing leaves the divisions of epidermal cells are controlled in such a way that under drought conditions, fewer stomata arise. This process is under the control of the mature leaves and their perception of water status. ABA is a key molecule also in regulating stomatal development (Chater et al. 2014).

6.4.2 Stimulation of Root Growth

The root system architecture belongs to the “hidden half” of plant biology. Plasticity of root growth has, for a long time, not been studied as extensively as the plasticity of shoot growth. This changed only recently (Chap. 7), at least partly fuelled by the expectation that major advances in sustainable agriculture may be achievable by breeding for relevant root traits.

A general shift in plant growth under water limitation is the increase in the root to shoot biomass ratio. This applies at least to situations of mild stress. More pronounced drying of the soil inhibits root growth as well, in part because of high soil impedance—that is, an increase in the force needed to penetrate the soil. Overall, the plasticity of the root system is even greater than that of the shoot. For instance, the surface area can vary more widely because of the tremendous influence of root hairs on the total surface. Mechanistically, the modulation of root system architecture in response to soil water is barely understood. Auxin gradients and cytokinins play a major role in controlling root morphology. ABA is known to influence the respective pathways.

6.5 Sensing of Water Status and Signal Transduction

As described above, a multitude of responses and acclimations is triggered by water deficit. Since the plant is part of the hydraulic soil–plant–air continuum and the cells are symplastically connected (with the exception of guard cells) (Steudle 2001), responses have to be coordinated in the entire organism. The stomatal aperture, for instance, has to be synchronised with the resistance of leaf and root tissue to water flow. Water shortage is translated into the stimulation of root growth. There is ample evidence for rapid responses of shoots to the root water status, and vice versa. Thus, long-distance signalling has to be in operation.

The most important endogenous signalling molecule mediating these responses is ABA. Its synthesis is rapidly and strongly up-regulated under drought stress (Fig. 6.17). Mutants lacking the ability to synthesise ABA are not able to withstand even a mild water deficit. ABA activates the majority of avoidance and tolerance mechanisms, including stomatal closure, the regulation of aquaporin abundance and activity, synthesis of protective proteins and osmoregulation. There is, however, also ABA-independent drought stress signalling. Furthermore, the rapid signalling of water status from the roots to the shoots, and vice versa, cannot be explained by ABA alone. ABA increases have frequently been detected in the xylem sap. However, grafting experiments with tomato and A. thaliana plants have demonstrated that leaves can respond normally to water deficit perceived by roots even when the roots are unable to synthesise ABA, so no ABA can travel to the shoot with the transpiration stream (Christmann et al. 2007).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig17_HTML.jpg — Fig. 6.17
Abscisic acid (ABA) synthesis is rapidly up-regulated upon dehydration of leaves. Upon water deficit, genes encoding the nine-*cis*-epoxycarotenoid dioxygenases (*NCED*s) are transcriptionally activated. NCEDs catalyse the rate-limiting step in ABA synthesis. This activation results in a rapid rise in ABA concentrations. a Time course of changes in *NCED* messenger RNA (mRNA) and NCED protein abundance, as well as ABA concentrations occurring in detached, wilting *Phaseolus vulgaris* leaves. b Western blot and northern blot analysis of NCED protein and *NCED* mRNA, respectively. (Qin and Zeevaart 1999)

ABA is an ancient molecule, detectable in all organisms with the exception of Archaea. Still, phylogenetic analysis of the main components of the core ABA signalling module (Fig. 6.20) suggests that ABA-dependent signalling apparently evolved in land plants (Hauser et al. 2011). Most drought stress avoidance and tolerance mechanisms are ABA dependent. Also, ABA triggers both rapid responses (within minutes), such as the closing of stomata, and responses that require changes in gene expression—that is, responses that depend on the interaction of transcription factors with cis elements in the promoters of stress tolerance genes and take hours to days. Three layers of ABA action can be differentiated: synthesis and transport, perception, and signal transduction (Hauser et al. 2011).

6.5.1 Sensing of Water Status

How a plant cell senses its water status and converts this into a signal has not been fully elucidated yet. Principally, two modes are discussed: the sensing of mechanical forces exerted on the plasma membrane by changes in turgor pressure or the sensing of osmotic potential by proteins sensitive to osmotic changes. The former could be mediated by mechanosensitive ion channels, the latter by osmosensitive kinases or other proteins able to transduce signals. Examples of both types of sensing are known from bacteria and yeast.

One current model assumes the sensing of a long-distance hydraulic signal (low water potential) by cells in the vasculature that would locally be transformed into a chemical signal—namely, ABA. Sensing of the water status and stimulated ABA synthesis are connected by a signal transduction pathway involving transient increases in cytosolic Ca²⁺ (Fig. 6.18) (Christmann et al. 2013). Recently, a first plant osmosensor may have been identified in A. thaliana (Yuan et al. 2014). Through a genetic screen for mutants with an impaired cytosolic Ca²⁺ response upon exposure to hyperosmolality, a Ca²⁺-permeable channel gated by hyperosmolality was found. When this channel (OSCA1) is defective, both rapid cellular responses and long-term whole-plant responses to osmotic stress are compromised. Plants can still respond to ABA normally, yet stomatal closure upon water deficit is impaired. This places OSCA1 upstream of ABA signalling and fulfils the criteria for the hypothetical osmosensor depicted in Fig. 6.18.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig18_HTML.png — Fig. 6.18
Model of hydraulic signalling: a long-distance hydraulic signal (decrease in water potential (Ψ_w)) travels through the plant in the xylem and is locally converted into a biochemical signal. The decrease in Ψ_w causes water flux along the Ψ_w gradient out of the parenchyma cells within, for example, the shoot vasculature, as shown here. The resulting decrease in turgor pressure (Ψ_p) and osmotic potential (Ψ_S) is sensed by an unidentified receptor, which triggers a signalling cascade (a transient increase in cytosolic Ca²⁺ and production of reactive oxygen species (ROS)). The signalling activates ABA biosynthesis. ABA synthesised in parenchyma cells is exported to bundle sheath cells and beyond to trigger acclimative responses. (Modified from Christmann et al. (2013))

6.5.2 ABA Signal Transduction

ABA biosynthesis is known to occur predominantly in vascular parenchyma cells of roots and shoots (Fig. 6.18). Both the responsible enzymes and the expression of the respective genes have been detected there. Plasma membrane–localised ABC-type transporters such as ABCG25 in A. thaliana can export ABA. Cells respond to ABA synthesised by the cell itself and to ABA taken up from the apoplast. Uptake of ABA into guard cells is mediated by another ABC-type transporter (ABCG40 in A. thaliana). Lack of its activity reduces the responsiveness of guard cells to ABA (Fig. 6.19).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig19_HTML.jpg — Fig. 6.19
Abscisic acid (ABA) import into guard cells is required for the normal ABA response, emphasising the importance of ABA uptake into guard cells and intracellular ABA sensing. Elevation of leaf temperature due to stomatal closure and a concomitant decrease in transpirational cooling can be detected after ABA treatment. The response is delayed in *atabcg40* plants defective in an ABA-importing ABC-type transporter. Less ABA reaches the inside of the guard cells per unit of time. The leaf temperature was monitored using an infrared thermal imaging camera after the addition of ABA. (Kang et al. 2010)

The earliest events in ABA signalling are mediated by a central regulatory module, which consists of three protein classes, the soluble ABA receptor PYR (PYRABACTIN RESISTANCE; also called RCAR, for REGULATORY COMPONENTS OF ABA RECEPTORS), protein phosphatases 2C (PP2C) and protein kinases (SnRK2s for SNF1-related protein kinases 2) (Fig. 6.20). Several isoforms of each of these proteins are encoded by plant genomes. This provides flexibility for the ABA signalling, which occurs in all kinds of cells and during all developmental stages. The signalling pathway formed by the three types of proteins is double negative. In the inactive state the protein SnRK2, which triggers the ABA responses, is inhibited by PP2C. Upon binding of ABA the receptor PYR interacts with PP2C and thereby inactivates it. This releases SnRK2 from the inhibition and enables the phosphorylation of several possible target proteins.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig20_HTML.png — Fig. 6.20
The core abscisic acid (ABA) signalling module. Protein phosphatases 2C (PP2Cs, *yellow*) inhibit protein kinases (SnRKs) (indicated by the *red symbol*) in the absence of ABA. ABA is bound by intracellular receptors (PYR dimers), which dissociate and form ABA receptor–PP2C complexes. This complex formation inhibits PP2Cs and allows activation of SnRKs. SnRKs then phosphorylate (indicated by *blue arrows*) target proteins. Several SnRK targets are known, of which only a subset are depicted here. ABA can activate both fast and slow responses because the SnRKs can modulate either ion channel activity or transcription factor activity, respectively. In addition, SnRKs can trigger downstream signalling events—for instance, by activating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (AtRbohF), which produce reactive oxygen species. (Hubbard et al. 2010)

The comparatively slow transcriptional responses to ABA are brought about by the phosphorylation of transcription factors such as AREB1 and AREB2. In the phosphorylated state these proteins are able to interact with ABA response elements (ABRE) in the promoters of ABA-responsive genes and to activate transcription. Target genes of these and related transcription factors encode, for instance, protective proteins such as LEA proteins and enzymes involved in compatible solute synthesis.

ABI5 is a transcription factor whose target genes maintain seed dormancy—one of the developmental processes under ABA control. Thus, the core signalling module is important for all types of ABA-dependent processes.

Rapid responses of guard cells are elicited by the activation of the anion channel SLAC1 and the inactivation of the inward-rectifying K ⁺ channel KAT1. The resulting depolarisation of the plasma membrane opens outward-rectifying K ⁺ channels, resulting in K⁺ efflux, turgor loss and stomatal closure (Figs. 6.13 and 6.14). SnRK2 also triggers additional signalling events such as the formation of ROS by NADPH oxidases (e.g. RbohF).

Besides ROS, other second messengers are involved in ABA signalling. Transient increases in cytosolic Ca ²⁺ concentrations, as a very common element of signalling cascades (Chap. 2), are observed in ABA-treated guard cells too. The central ABA signalling module (Fig. 6.20) is not Ca²⁺ dependent. Instead, Ca²⁺ signalling modulates targets of the core pathway. ROS production stimulates a cytosolic Ca²⁺ increase which, via the action of Ca²⁺-dependent protein kinases, affects the activity of anion channels such as SLAC1. It is conceivable that the necessary integration of various environmental and physiological cues in guard cells, which is essential for a plant responding to many simultaneously changing aspects of its natural environment, is at least in part achieved by the convergence of several signalling pathways on the anion channels as central control points for the plasma membrane potential. Anion channel phosphorylation in multiple sites by different kinases controlled by distinct signalling pathways could provide a mechanism for such signal integration (Kollist et al. 2014).

6.5.3 ABA-Independent Signalling

ABA is clearly the central stress hormone controlling water deficit responses. Still, in mutant plants unable to respond to ABA, there can be induction of drought stress-responsive genes. This demonstrates the existence of signalling pathways that do not require ABA. A known pathway activates transcription factors of the CBF/DREB class, which are involved in ABA-independent signalling during cold acclimation (Chap. 4). Some of these proteins trigger dehydration responses instead. The promoters of their target genes contain a cis element called the dehydration responsive element (DRE) or C-repeat.

6.6 Photosynthesis Variants with Improved Water Use Efficiency

Plants are dependent on the uptake of CO₂ through stomata, which inevitably results in evaporation of H₂O. Two major variants of photosynthesis have evolved that reduce the amount of water vapour lost per unit of carbon fixed. In other words, they increase the water use efficiency—that is, the ratio of dry weight gained (= growth) to water lost. In regular C ₃ photosynthesis, about 500 g of H₂O is spent per gram of carbon assimilated. The cost is reduced to about 250 g of H₂O per gram of carbon in plants displaying C ₄ photosynthesis and to only 50–100 g of H₂O in CAM plants (Table 6.3). Both mechanisms share a first fixation of CO₂ by the enzyme phosphoenolpyruvate carboxylase (PEP carboxylase) prior to a second ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)–dependent fixation of CO₂ after its release through decarboxylation of the storage molecule malate. The competitive advantage of higher water use efficiency is evident from the fact that plants showing these photosynthesis variants are predominantly found in arid and dry regions of the world (Chap. 10).

Table 6.3

Water use efficiency, photosynthesis and biomass production of C₃, C₄ and CAM plants. Crassulacean acid metabolism (CAM) plants are superior to other photosynthetic types in their water use efficiency^a, but their photosynthetic rates and growth rates are much lower (Lüttge et al. 1994)

Type of photosynthesis	C₃	C₄	CAM
Water use efficiency (g water/g C)	450–950	250–350	18–100 (night-time)
Water use efficiency (g water/g C)	450–950	250–350	150–600 (daytime)
Maximum rate of net photosynthesis (μmol CO₂ m⁻² s⁻¹)	9–25	25–50	0.6–8
Growth (g biomass m⁻² day⁻¹)	50–200	400–500	1.5–1.8

^aWater use efficiency is defined as the ratio of dry weight gained (= growth) to water lost

6.6.1 C₄ Photosynthesis

In every plant, CO₂ is fixed by RubisCO. This enzyme, which represents by far the most abundant protein on Earth, catalyses the first reaction of the Calvin cycle between an activated pentose phosphate, ribulose-1,5-bisphosphate and CO₂ (the carboxylase activity), yielding two molecules of 3-phosphoglycerate—a molecule with three C atoms (hence the term C ₃ photosynthesis). In conditions of a vast excess of O₂ versus CO₂, RubisCO also catalyses the reaction of ribulose-1,5-bisphosphate with O₂ (the oxygenase activity), yielding one molecule of 3-phosphoglycerate and one molecule of 2-phosphoglycolate. This side reaction poses a problem, as 2-phosphoglycolate is a useless toxic metabolite that should not accumulate. 2-Phosphoglycolate is converted to 3-phosphoglycerate via photorespiration, a pathway that requires the metabolic activity of three organelles—namely, peroxisomes and mitochondria, besides chloroplasts where 2-phosphoglycolate is produced (see plant physiology and plant biochemistry textbooks). Of the carbon in 2-phosphoglycolate, 75% is returned to the Calvin cycle as 3-phosphoglycerate.

The efficiency of ribulose-1,5-bisphosphate carboxylation is about 100-fold higher than that of the oxygenation. Thus, only gradually with the accumulation of oxygen in the atmosphere did this side reaction of RubisCO become relevant. RubisCO evolved in an atmosphere that was essentially devoid of molecular oxygen.

Furthermore, it is ecologically important that the carboxylation to oxygenation ratio of RubisCO is influenced by temperature. With higher temperature the oxygenase activity becomes more relevant because RubisCO specificity decreases. Therefore, the need for photorespiration, which negatively affects photosynthetic efficiency in plants with C₃ photosynthesis, grows with increasing temperatures.

At a time about 30 million years ago, when atmospheric CO₂ reached a critically low level, C₄ photosynthesis arose. It has evolved independently many times since then (>60 times according to current counts) in multiple plant families. Through a series of anatomical and biochemical modifications, C₄ photosynthesis achieves a higher concentration of CO₂ in the vicinity of RubisCO, thereby effectively suppressing the oxygenase activity. This results in higher photosynthetic efficiency under conditions that promote photorespiration (low CO₂, high temperature). Typically, two consecutive fixations of CO₂ occur in separate cell types. In mesophyll cells, which in C₄ plants often surround the vascular bundles (including the bundle sheath cells) in a circular arrangement called the Kranz anatomy, CO₂ is fixed by PEP carboxylase and not by RubisCO, as in C₃ plants. In C₄ plants, RubisCO activity is restricted to the bundle sheath cells which, in contrast to C₃ plants, are more prominent and contain chloroplasts. Fixation of CO₂ as bicarbonate by PEP carboxylase (which does not catalyse a reaction with O₂) produces a C₄ acid, which is then shuttled into the bundle sheath cells where decarboxylation releases CO₂. This CO₂ is utilised by RubisCO in the Calvin cycle (Fig. 6.21). Concentration of CO₂ in the vicinity of RubisCO by primary CO₂ fixation via PEP carboxylase and shuttling of carbon into bundle sheath cells can be seen as the core of C₄ photosynthesis. Variations between plant species exist with respect to the type of C₄ acid (e.g. malate or aspartate) and the nature of the decarboxylating enzymes (malic enzyme in Fig. 6.21).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig21_HTML.jpg — Fig. 6.21
The C₄ photosynthesis core mechanism. The *green boxes* represent the chloroplasts. *Blue dots* represent active transport steps. CA carbonic anhydrase, *MDH* malate dehydrogenase, ME malic enzyme, *PEPCase* phosphoenolpyruvate carboxylase, *PPdK* pyruvate orthophosphate dikinase, *PEP* phosphoenolpyruvate, *OAA* oxaloacetate, *RuBP* ribulose-1,5-bisphosphate, *3-PGA* 3-phosphoglycerate. (Modified from Langdale (2011))

The CO ₂ pumping allows RubisCO to operate at near substrate saturation. This offers the potential for very high photosynthetic rates. Furthermore, it enables C₄ plants to reduce stomatal conductance, which decreases water loss and improves water use efficiency. An additional advantage is the lower nitrogen requirement of C₄ plants. Much less investment in RubisCO protein is needed. Higher efficiency of photosynthesis and lower demand for water explain why, today, many biomes—especially in the tropics and subtropics (such as the African savannas)—are dominated by C₄ plants. Of today’s vascular plant species, 3% use C₄ photosynthesis. They account for about 25% of total terrestrial photosynthesis. Nevertheless, the distribution of C₄ plants clearly shows that by no means all biomes favour C₄ photosynthesis (Chap. 12, Sect. 12.1). The primary CO₂ fixation consumes extra metabolic energy because the substrate PEP has to be regenerated from pyruvate, a reaction that consumes adenosine triphosphate (ATP). Thus, C₄ plants have lower quantum use efficiency than C₃ plants under conditions where photorespiration is low (low light, low temperature).

6.6.2 Evolution of C₄ Photosynthesis

C₄ photosynthesis is characterised by a suite of distinct anatomical, morphological, physiological and biochemical features. Therefore, it is, at first sight, surprising that C₄ photosynthesis has evolved independently so many times from ancestral C₃ photosynthesis. Owing to a large body of research work, we can today describe C₄ photosynthesis as an excellent example of how the evolution of a key adaptation can be understood in an ecological context (Christin and Osborne 2014). Conceptual models have been proposed as to how C₄ photosynthesis developed (Fig. 6.22). Evolution along this path is plausible because for every step, distinct selective advantages can be inferred and factors priming C₃ lineages for the evolution of these steps can be identified. Moreover, some of the steps can be seen in extant C ₃ –C ₄ intermediate species (e.g. in the genus Flaveria), which represent different stages on the evolutionary trajectory towards C₄ photosynthesis.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig22_HTML.png — Fig. 6.22
Model of the stepwise evolution of C₄ photosynthesis from C₃ photosynthesis (Gowik and Westhoff 2011); see also a similar model in Sage et al. (2012)

All enzymes employed in C₄ photosynthesis are of ancient bacterial origin. A first preconditioning step towards C₄ photosynthesis was the duplication of respective enzyme-coding genes or of whole genomes. The existence of more than one gene copy enabled diversification with respect to localisation, timing and strength of expression. This occurred predominantly via changes in promoter sequences. Furthermore, kinetic properties could change through alterations in coding sequences (right arrow in Fig. 6.22). For example, PEP carboxylases of C₄ plants are less inhibited by malate. This is functionally important, as CO₂ fixation by PEP carboxylases has to function in the presence of high malate concentrations (Fig. 6.21). The PEP carboxylase genes in C₄ plants show signs of strong positive selection in their sequences, meaning there is evidence that amino acid changes affecting the kinetic properties of the encoded enzymes were selected during the evolution of C₄ photosynthesis.

The efficient exchange of metabolites between mesophyll and bundle sheath cells is promoted by high vein density, as this reduces the distances between the two cell types. The high vein density typical of many C₄ lineages offers advantages for C₃ plants too. For example, there are more pathways for water transport into and through the leaves. This could improve drought tolerance and photosynthetic rates in arid, high-light environments.

The higher proportion of leaf volume occupied by bundle sheath cells with only a few chloroplasts and therefore low photosynthetic capacity could have exerted selective pressure to increase the number of chloroplasts and other organelles in these cells. Related to the higher metabolic activity of bundle sheath cells in C₄ plants is the photorespiratory CO ₂ pump found in extant C ₃–C ₄ intermediates. Restriction of the glycine decarboxylase activity (for details of the photorespiration pathway, see plant biochemistry or plant physiology textbooks) to mitochondria of bundle sheath cells forces the processing of all photorespiratory glycine in these cells (sometimes this is referred to as the “C₂ cycle”). The decarboxylation releases CO₂ at a site more distant from the leaf surface, thus improving the chances for refixation by RubisCO, whose oxygenation activity is suppressed by the extra CO₂. Molecularly the photorespiratory CO₂ pump can arise easily. It takes only two genes encoding a subunit of the glycine decarboxylase complex with expression restricted to either mesophyll or bundle sheath cells by the right cis elements in the promoters. Loss of function of the mesophyll-expressed version would then result in glycine decarboxylase activity only in the bundle sheath cells and thereby establish the CO₂ pump.

Conversely, the levels of carbonic anhydrase and PEP carboxylase had to massively increase in the cytosol of the mesophyll cells. The C₄ cycle is then completed through the spatial separation of the two carboxylase reactions, PEP carboxylase in the mesophyll and RubisCO in the bundle sheath cells. In the course of C ₄ cycle optimisation, many other metabolic changes have evolved that can mostly be explained by changes in transcriptional regulation too. A recent modelling of the biochemical fitness landscape between C₃ and C₄ photosynthesis—based on kinetic parameters of enzymes, gas exchange rates, etc.—demonstrated that indeed every step along the different evolutionary trajectories from C₃ to C₄ photosynthesis is associated with a fitness gain (Heckmann et al. 2013). This leaves the question as to why not all angiosperm lineages have evolved C₄ photosynthesis. A possible explanation could be that certain potentiating factors such as high vein density are not present in all lineages.

6.6.3 Crassulacean Acid Metabolism

CAM represents an important metabolic adaptation to water scarcity. It is characterised by highly efficient water use, coming at the expense of slow growth. CAM photosynthesis has been found in at least 36 taxonomically diverse plant families and has therefore most probably also evolved multiple times independently, like C₄ photosynthesis. About 6% of all flowering plant species are CAM plants. They thrive mostly in dry and hot regions, or are confronted with water scarcity because they live as epiphytes—for instance, orchids on trees in tropical forests. CAM plants restrict water loss by a reversal of stomatal regulation in comparison with non-CAM plants. Stomata are closed during the day and opened at night when the air is much cooler and more humid; that is, when the vapour pressure deficit between leaf and air is lower (Fig. 6.23). CAM photosynthesis usually is combined with several anatomical and morphological features that further minimise water loss. They include highly efficient sealing of above-ground surfaces by thick cuticles, low surface to volume ratios, succulence (i.e. large cells and vacuoles with enhanced water storage capacity) and lower stomatal density (Cushman 2001; Silvera et al. 2010).

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig23_HTML.png — Fig. 6.23
Key steps in crassulacean acid metabolism (CAM) photosynthesis. (Modified from Borland et al. (2014))

Usually the CAM metabolism is divided into four phases (Fig. 6.24). Because of the nocturnal stomatal conductance, CO₂ is taken up in the dark. Primary carbon fixation is catalysed by PEP carboxylase but, in contrast to C₄ photosynthesis, primary and secondary carbon fixations are separated temporally, not spatially. Phosphoenolpyruvate reacts with HCO₃ ⁻ to yield oxaloacetate. Oxaloacetate is then reduced in the cytosol to malate by NAD(P)-dependent malate dehydrogenase and stored in the vacuole as the protonated form, malic acid. As a result the vacuoles of CAM plants strongly acidify (up to pH 3) (by the end of phase I). Early in the light period, stomata may still be open and some CO₂ fixation by RubisCO can occur (in phase II). Later during the day, malate is released from the vacuole (de-acidification) and converted by the NADP-dependent malic enzyme or PEP carboxykinase into CO₂, reduction equivalents (NADPH) and pyruvate. CO₂ is assimilated by RubisCO in the reductive photosynthetic carbon cycle (Calvin cycle)—that is, the regular C₃ photosynthesis path. However, in contrast to C₃ plants, the RubisCO operates at saturating CO₂ concentrations and oxygenation of ribulose-1,5-bisphosphate is strongly reduced (end of phase III). Phase IV comprises the second transition when organic acid stores are depleted and stomata open again if environmental conditions permit. Liberated C₃ acids are converted into storage carbohydrates. The primary CO₂ acceptor, PEP, is synthesised from storage carbohydrates. One of the main enzymes catalysing this reaction is pyruvate orthophosphate dikinase (PPdK). Typical diurnal rhythms of gas exchange, as well as malic acid and starch concentration, are shown in Fig. 6.24. In contrast to C₃ and C₄ plants, the rate of CO₂ uptake by CAM plants is limited by mesophyll processes—such as the provision of acceptor molecules for carboxylation derived from storage carbohydrates or vacuolar storage capacity—and not by stomatal conductance. The need for malic acid storage is one additional reason for the frequent association of CAM photosynthesis with succulence.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig24_HTML.png — Fig. 6.24
Typical diurnal rhythms of a crassulacean acid metabolism (CAM) plant. a Gas exchange curve (CO₂ uptake rate and stomatal resistance = stomatal conductance⁻¹) (*upper panel*) and changes in the concentrations of the typical metabolites starch and malate (*lower panel*). The gas exchange shows phase I: nocturnal CO₂ fixation; phase II: dawn, with the start of CO₂ fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO); phase III: assimilation of the internally released CO₂ by RubisCO with the stomata closed; and phase IV: stomata begin to open after the internal CO₂ has been depleted and daytime temperatures are dropping. In b the time courses of the different reactions are indicated by different shades of *blue*. a Lüttge et al. (1994); b Cushman and Bohnert (1999)

The regulation of PEP carboxylase activity in CAM plants represents an illustrative example of the importance of the biological clock and controlled day/night cycles in adaptation (Chap. 2). During the day, the refixation of CO₂ by PEP carboxylase has to be down-regulated in order to prevent competition with RubisCO for the substrate and a concomitant futile cycle of malate synthesis and decarboxylation. Regulation occurs via phosphorylation of the enzyme by a kinase whose activity oscillates with a circadian rhythm. When the N-terminus of the enzyme is phosphorylated, PEP carboxylase is less feedback inhibited by the end product malate and can thereby provide sustained primary CO₂ fixation during the night. The non-phosphorylated form is inhibited by about ten times lower malate concentrations. Due to these properties, a rhythmic alternation between a more active “night form” and a less active “day form” of the enzyme is achieved through circadian transcriptional regulation of the responsible kinase, PEPC kinase. The gene is more actively transcribed during the night, resulting in the presence of the more active phosphorylated form of PEPC during nighttime. Other processes such as the provision of PEP are also under circadian control.

CAM shows pronounced physiological plasticity and ecological diversity. Depending on the environmental conditions, different manifestations of CAM are distinguished. During long periods of drought, stomata remain continuously closed, even at night. Fixation and assimilation of CO₂ are limited to CO₂ generated internally in the plant tissue by respiratory processes. This state is called CAM idling. The photosynthetic assimilation of internal CO₂ provides some protection against photoinhibition. However, the low internal CO₂ content, combined with the usually extremely high radiation, is not sufficient to allow a substantial flow of electrons and full relief of the photosystems. Consequently, oxygen radicals are formed and oxidative stress occurs. Therefore, CAM idling is of only limited use, as it cannot be sustained for extended periods of time.

Many bromeliads show diurnal CAM metabolism, even though no or very little nocturnal CO₂ fixation by PEP carboxylase occurs. This is called CAM cycling or weak CAM (meaning an evolutionarily early form of CAM). An extreme example of CAM cycling occurs in usually submerged water plants such as Isoëtes howellii, a lycophyte. The epidermis of this plant, of course, has no stomata, and the plant does not experience water deficit stress (Keeley 1998). The reason for the CO₂ fixation during the night is assumed to be the lack of CO₂ in the usually acidic waters where Isoëtes grows. CAM cycling has also been found in flowering aquatic plants (e.g. the genus Sagittaria). Latent CAM refers to high but not diurnally cycling organic acid concentrations and is thought to represent a C₃–CAM intermediate stage.

CAM can also be facultative—that is, induced upon exposure to low water availability—in species that in the presence of sufficient water supply show normal C₃ photosynthesis. This represents an effective acclimation to salt or drought stress conditions and has been most intensively studied in the common ice plant M. crystallinum (Cushman 2001). CAM induction can be irreversible, as in M. crystallinum, or reversible. In some facultative CAM plants, only certain leaves or developmental stages display CAM photosynthesis. The succulent stem of Frerea indica (an asclepiad) performs CAM, while the green leaves—at least in rainy periods—photosynthesise in the C₃ mode. In tropical Clusia species with opposite leaves at a node, one leaf may perform C₃ photosynthesis while the opposite leaf uses CAM (Lüttge 1987).

The magnitude of the transition is strongly influenced by the degree of water scarcity and other environmental factors, including light intensity, temperature and humidity. This ecophysiological plasticity allows the C₃–CAM intermediates optimal acclimation of their photosynthetic capacity to the prevailing conditions, making effective use of the humid season or of sporadic precipitation. The shift to CAM photosynthesis in response to drought or salinity involves transcriptional up-regulation of many genes encoding enzymes involved in CAM. The first example was a PEP carboxylase gene in M. crystallinum. More recent transcriptome studies have revealed changes in >2000 genes during the transition from C₃ to CAM photosynthesis (Cushman et al. 2008).

Whether a facultative CAM plant, or a part of the plant, follows C₃ photosynthesis or CAM photosynthesis can be determined using the δ ¹³C value. Of the carbon dioxide in the air, 98.89% consist of the ¹²C isotope and 1.11% consist of ¹³C. The content of the radioactive isotope ¹⁴C is, in comparison with those values, negligible (10⁻¹⁰%). RubisCO consumes CO₂ as a substrate and discriminates more strongly between ¹²C and ¹³C than does PEP carboxylase, which uses HCO₃ ⁻ instead of CO₂. Carbon assimilated only by RubisCO thus contains less ¹³C than that fixed first by PEPC. The change of ¹³C in the biomass therefore allows determination of the mode of CO₂ fixation. The ratio of ¹³C to ¹²C is determined by mass spectrometry and calculated with the following formula:

${\delta}^{13}\left[{\mbox{\fontencoding{U}\fontfamily{wasy}\selectfont\char104}} \right]=\left[\frac{{}{}^{13}\mathrm{C}/{}{}^{12}\mathrm{C}\kern0.5em \mathrm{of}\ \mathrm{sample}}{{}{}^{13}\mathrm{C}/{}{}^{12}\mathrm{C}\kern0.5em \mathrm{of}\ \mathrm{standard}}-1\right]\times 1000$

The standard is a defined limestone. The δ¹³C values of C₃ plants are around −28‰; those of C₄ plants are around −14‰; and those of CAM plants with predominantly nocturnal CO₂ fixation are between −10 and −20‰, and for daytime CO₂ fixation, between −25 and −34‰.

6.6.4 Evolution of Crassulacean Acid Metabolism Photosynthesis

The physiological plasticity of CAM corresponds to its evolutionary diversity. CAM manifestation is strongly influenced by the history of a species and its habitat context (Silvera et al. 2010). CAM is taxonomically more broadly distributed than C₄ photosynthesis and is most likely evolutionarily older. The presence of CAM in ancient groups such as the Isoëtes (see I. howellii above) suggests a first appearance of CAM already in the Triassic (250–200 million years ago). Further CAM evolution was then probably driven by selection for increased carbon gain and better water use efficiency after the global reduction in atmospheric CO₂ concentration about 30 million years ago during the Oligocene (see evolution of C₄ photosynthesis, Sect. 6.6.2). CAM has evolved many times independently and to varying degrees. The extent of CAM manifestation shows a positive correlation with the dryness of the site; that is, the stronger the water scarcity of a habitat, the higher the likelihood of full CAM expression in the plants populating it.

Several characteristics of CAM can be distinguished, as described above (Borland et al. 2014). Accordingly, CAM requires a number of evolutionary changes in basal mechanisms that principally are present in all higher plants (Fig. 6.25):

First, a reversal of stomatal regulation enables nocturnal CO₂ uptake. The control of the stomatal aperture by light has to be overridden by other control mechanisms. One factor could be the low internal leaf CO₂ partial pressure at night, due to the activity of PEP carboxylase
Second, diurnal fluctuations in organic acids and (reciprocally) in storage compounds and soluble sugars, plus respective transport activities, are established (e.g. malate into the vacuole and malic acid out of the vacuole)
Third, key elements of CAM photosynthesis, e.g. carboanhydrase and PEPC, as well as decarboxylating enzymes—are more strongly expressed. Prerequisites here are the duplication and diversification of genes encoding the respective enzymes. As in plants with C₄ photosynthesis, there are CAM plant–specific isoforms of PEP carboxylase with very high leaf expression
Fourth, enhanced gluconeogenic and glycolytic activities supply substrates for carboxylation and decarboxylation
The fifth element is leaf succulence. A clear correlation exists between the degree of leaf succulence and the strength of CAM. Plants with thicker leaves show lower δ¹³C values, which is indicative of stronger CAM (see the range of δ¹³C values in Table 6.3). This is explained not only by the greater storage capacity of larger cells (with vacuoles taking up 90–95% of the volume) but also by the tight packing of cells in succulent tissues, which restricts the intercellular gas space and thereby the gas exchange rates, and consequently limits C₃ photosynthesis during phases II and IV
Finally, the sixth key mechanism is the circadian clock control over CO₂ fixation. Comparative studies with four Clusia species (one C₃ species, two C₃–CAM intermediates and one strong, constitutive CAM species) have revealed an association of the circadian control of PEPCK transcript abundance with CAM strength—that is, with day/night changes in malate and soluble sugar content

As indicated in Fig. 6.25, all of these mechanisms can vary in their extent along a continuum of CAM manifestations.

/epubstore/S/E-D-Schulze/Plant-Ecology/OEBPS/images/72100_2_En_6_Chapter/72100_2_En_6_Fig25_HTML.png — Fig. 6.25
Evolutionary changes required for crassulacean acid metabolism (CAM) photosynthesis. (After Silvera et al. (2010))

6.7 Summary

The strict association between CO₂ uptake and water loss can be referred to as the central dilemma of plants: dying of thirst or dying of hunger?
Life requires liquid water and is thus dependent on particular physico-chemical properties that result from the dipole nature of the water molecule (the so-called anomalies of water).
The water potential (Ψ_w) is a measure of the thermodynamic state of water in any system and is given in the dimension of pressure. Figuratively speaking, it is a measure of the energy required to remove water molecules from any water-containing system. The potential of pure water under standard conditions of pressure and temperature is defined as zero and used as a reference. Any system that requires more energy to remove water from has a negative water potential. Thus, water moves towards the lower (more negative) potential.
The water potential of a solution, and correspondingly that of a cell, is influenced by three major components: the concentration of solutes, termed the osmotic potential (Ψ_s), the pressure potential (Ψ_p) and gravity (Ψ_g). When considering water potential beyond the cellular level or in cells and other structures in an at least partially dehydrated state, the matrix potential (Ψ_m) is often included as a fourth component.
Water movement in a plant follows gradients in potential. It is always passive, as no mechanical pump is in operation. The plant, however, actively influences the direction and the steepness of these gradients.
In order to take up water from the soil solution, the water potential of root cells has to be more negative than that of the surrounding soil. Plant cells can lower their water potential by osmotic adjustment. Increasing the concentration of solutes makes the osmotic potential (Ψ_s), and thereby the water potential of the cell, more negative.
Three pathways for the flux of water through a tissue exist: apoplastic, symplastic, and transcellular. Uptake into the symplasm and transcellular flux both require membrane passages. The resistance of biomembranes for water flux is lowered by water channels, the aquaporins. Plants possess a large number of aquaporin isoforms with distinct expression patterns and subcellular localisations. The principal ways of controlling aquaporin-dependent water flow are modulation of aquaporin abundance and permeability (= gating).
The multitude of aquaporin isoforms in a plant and the many ways of regulating them (e.g. transcription, subcellular localisation, phosphorylation, ubiquitination) provide the means to acclimate to fluctuations in water availability and demand by adjusting resistance for water flow across tissues, as well as into and out of cells and cellular compartments.
Principally two different strategies to cope with water scarcity can be distinguished: avoidance and tolerance. However, these categories merely represent the extreme poles of a continuum of responses that are dependent on the plant and the severity of the drought stress. Avoidance refers to a balancing of water uptake and water loss that maintains the water status (in isohydric plant species). Tolerance mechanisms help a plant endure a moderate lowering of the water potential (in anisohydric plant species).
Osmotic adjustment is achieved by the accumulation of “compatible solutes” or “osmolytes”—organic low molecular weight compounds of different chemical classes. The protective function goes beyond the osmotic effect. Some compatible solutes can scavenge reactive oxygen species; others stabilise proteins and membranes.
Protective proteins accumulate under drought stress to shield cellular structures from damage caused by water loss. Among them are the late embryogenesis abundant (LEA) proteins, which are also formed during seed maturation as part of a plant’s regular developmental programme.
An extreme form of drought tolerance is shown by poikilohydric plants, which withstand near complete tissue dehydration (desiccation). Desiccation tolerance represents an adaptation to extreme environmental conditions. It involves a state of dormancy characterised by massive accumulation of protective proteins.
Most of the transpirational water loss occurs via the stomata. Therefore, control of the stomatal aperture plays a key role in a plant’s response to water deficit and in drought stress tolerance. Several internal and environmental cues (e.g. water status, humidity, CO₂ concentration) are integrated by guard cells (which form the stomatal pore) in order to optimally adjust the stomatal aperture for any given physiological situation.
The stomatal aperture is nearly linearly correlated with guard cell turgor pressure. Thus, the concentration of solutes in guard cells determines stomatal conductance. Changes in K⁺ and Cl⁻ concentrations are brought about by ion channel–mediated exchange between guard cells and the surrounding apoplast, as well as between guard cell vacuoles and the cytosol. Control over stomatal movement is exerted by control over these ion fluxes.
An integral part of a plant’s acclimation to water deficit caused by drought, and essentially an avoidance strategy, is the reduction of leaf area relative to biomass; that is, a trade-off exists between stress tolerance and maintained growth. A second acclimation of growth under conditions of water scarcity is the stimulation of root growth in order to improve the water uptake capacity and to access additional water resources. Thus, a general shift in plant growth under water limitation is the increase in the root to shoot biomass ratio.
Growth control is regulated at the level of cell division and cell expansion.
Drought responses have to be coordinated in the entire organism, requiring long-distance signalling between organs.
The phytohormone abscisic acid (ABA) activates the majority of avoidance and tolerance mechanisms, including stomatal closure, the regulation of aquaporin abundance and activity, synthesis of protective proteins and osmoregulation.
Three layers of ABA action can be differentiated: synthesis and transport, perception, and signal transduction.
The earliest events in ABA signalling are mediated by a central regulatory module, which consists of three protein classes: receptors, protein phosphatases and protein kinases. This module triggers rapid responses—such as the activation and inactivation of ion channels—and slower responses at the transcriptional level.
Two major variants of photosynthesis have evolved that increase water use efficiency—that is, they reduce the amount of water vapour lost per unit of carbon fixed: C₄ photosynthesis and Crassulacean Acid Metabolism (CAM).
C₄ photosynthesis and CAM employ a first fixation of CO₂ (as bicarbonate) by PEP carboxylase before CO₂ is fixed a second time by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). In C₄ plants these CO₂ fixations are separated spatially; in CAM plants they are separated temporally.
Both variants achieve a higher CO₂ concentration in the vicinity of RubisCO. This suppresses the oxygenase activity of RubisCO and thereby the need for photorespiration.
C₄ plants can achieve efficient photosynthesis with a reduced stomatal aperture, which limits water loss. Provision of the substrate for the first CO₂ fixation requires extra energy. Taken together, these restrict the natural occurrence of C₄ plants essentially to environments characterised by water scarcity, high light and high temperature.
CAM plants reduce water loss even more. They open stomata at night so that less transpiration occurs. CO₂ is taken up and stored after fixation as malate in the vacuole for later use by RubisCO.
C₄ photosynthesis and CAM have evolved many times independently in different plant lineages. Models have been developed that describe the stepwise evolution of these major adaptations to arid environments.

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6. Water Deficiency (Drought)