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Recent Advances in Understanding the Regulation of Whole-Plant Growth Inhibition by Salinity, Drought and Colloid Stress
Recent Advances in Understanding the Regulation of Whole-Plant Growth Inhibition by Salinity, Drought and Colloid Stress
PETER M. NEUMANN1
Department of Environmental, Water and Agricultural Engineering, Technion Israel Institute of Technology, Haifa, Israel
I. II. III. IV.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plant Growth and Survival During Moderate and Severe Salinity Stress . . . Plant Growth During Moderate Water Stress Episodes . . . . . . . . . . . . . . . . . . . . . Whole-Plant Water Availability and Growth Can also be Limited by Colloid Stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT This chapter presents an eclectic perspective, based largely on research findings from our laboratory on biochemical and biophysical mechanisms involved in growth inhibition by water deficits, as caused by salinity (Section II), drought (Section III) and recently reported plant interactions with aqueous colloids found in soil solutions or xylem sap (Section IV). Much attention in each section is given to the roles of plant cell walls in regulating whole-plant growth inhibition under stressful conditions. One conclusion is that ongoing research into genomic and epigenetic changes that participate in the regulation of cell wall changes, cellular antioxidant status and plant hydraulics may provide new approaches for limiting the plant growth inhibition or mortality associated with salinity and drought. In Section IV, it is concluded that the ‘colloid stress’ that results from the inhibitory effects on water transport of physical
1
Corresponding author: E-mail: [email protected]
Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00002-3
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interactions between plant cell walls and environmental or internal colloids is a novel stress-factor that can affect plant water relations and further limit plant ability to resist salinity and drought.
I. INTRODUCTION Abiotic stress can be defined as an adverse situation resulting from plant exposure to sub- or supra-optimal levels of environmental inputs such as water, light, temperature or solutes. Abiotic stress resulting from water deficits or excessive salinity generally leads to reductions in photosynthesis, transpiration and growth. Such stresses are a major, world-wide-factor limiting the ability of mankind to produce adequate supplies of plant food, fibre and fuel for an expanding world population. Ongoing advances in botanical research into plant responses to abiotic stresses, in addition to increasing basic knowledge, may facilitate advances in our practical ability to maintain and increase plant productivity in stressful agricultural or natural environments. Botany, or that branch of biology that concerns the scientific study of plant life, has evolved rapidly since Watson and Crick (1953) published their seminal paper on DNA structure. The genomic era they signalled has been characterised by the introduction of increasingly sophisticated analytical technologies that now facilitate ‘deep’ genomic, transcriptomic, proteomic and metabolomic investigations. Most recently, there has been increasing interest in hereditable epigenetic changes involving DNA methylation, histone-modification and microRNA. These developments in cell molecular biology are leading to a post-genomic era where systems biology seeks to integrate understanding of the processes regulating metabolism into holistic models of development, albeit mainly at the cellular level. Parallel advances in research into transport and communication processes that are associated with development at the level of tissues, organs and whole organisms, are also needed for fuller understanding and more successful modifications of whole-plant responses to environmental change. An important area in which further study is certainly needed is the extracellular (apoplastic) region of plants, that is, plant cell walls, xylem and environmental boundary layers at root and leaf surfaces. Each of these can make direct ‘post-genomic’ contributions to the regulation of whole-plant growth and development under optimal or stressful conditions. An example of apoplastic changes that may regulate plant development is given by what happens as tension, that is, negative hydrostatic pressure, increases in the xylem water columns of plants facing water deficits. When critical tensions are reached, the formation of gaseous embolisms may disrupt xylem hydraulic continuity and thereby restrict essential xylem-transport of soil derived
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water and solutes to the shoot. Such physical reductions in water transport can then adversely affect leaf functioning and hence, plant growth and development (McDowell et al., 2008; Sperry et al., 2002). Much of the emphasis in this chapter is on apoplastic changes that are involved in the regulation of cell, organ and whole-plant growth responses to salinity and water stress. Such stresses can be moderate, that is, non-lethal, or extreme, that is, lethal. A central theme is that the molecular changes induced in plant cells by moderate environmental stresses will usually lead to rapid decrease in rates of growth of tissues and organs, be they roots, stems, leaves, flowers, fruits or seeds. Stress-induced changes in plant growth rates, tissue mechanics and phenology can therefore provide integrated reflections of underlying molecular changes (Hauben et al., 2009; Neumann, 1997; Nicotra and Davidson, 2010; Uyttewaal et al., 2010). The practical consequences of stress-induced decreases in the growth of plants can be viewed in different ways. For example, limiting new leaf area production under water stress not only will limit plant potential for photosynthesis but will also limit overall leaf transpiration. It may therefore extend the availability of limited soil water reserves and hence plant ability to survive long enough to produce (a limited amount of) seeds. Albeit, in an agricultural context and in the viewpoint of this chapter, stress-induced reductions in growth rates of crop plants will generally be associated with unwanted reductions in economic yields. Thus, stress acclimation that involves growth inhibition and associated increases in plant survival may still be viewed as a hindrance by farmers. In order to review the plant growth reductions induced by environmental stresses, it is necessary to first consider what exactly is meant by the term growth. Growth can be defined as an irreversible increase in size. In plants, vegetative and reproductive growth is always based on the expansion of young daughter cells produced by ongoing meristematic divisions. Growth is a prerequisite for plant development, and its regulation may occur via changes in the cell division cycle and in rates or duration of cell expansion (cf. Lu and Neumann, 1998; Skirycx and Inze, 2010). Note, however, that even cell division is dependent on a limited amount of prior cell expansion. The processes involved in the cell expansion that is primarily responsible for size increases at tissue, organ and whole-plant levels can be conveniently described by the Lockhart (1965) model equations. The first equation states that relative rates of cell expansion growth (RGR) are limited by hydraulic factors which control the entry of water required for cell volume increases. One such hydraulic factor is the maintenance of cytoplasmic water potential (Cc) at a level which is more negative than that of the external water source (Co). The resultant water potential gradient provides the driving force for
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water uptake and resultant flow is in turn, modulated by an additional factor, that is, the hydraulic conductivity, L, of the water conducting pathways leading into the cytoplasm. RGR ¼ LðCo Cc Þ
ð1Þ
The second Lockhart equation indicates that relative rates of cell growth are also co-regulated by interactions between cell turgor pressure (P), the mechanical extensibility of the cell walls (m) and the minimum turgor pressure required to start cell expansion, that is, the cell wall yield threshold (Y). RGR ¼ mðP Y Þ
ð2Þ
An often overlooked but important point is that each of the cellular parameters involved (Cc, L, P, m, Y) and hence cell growth itself, can be metabolically regulated. For example, L can be influenced by the opening or closure of aquaporin water channels in the membranes (e.g., Lu and Neumann, 1999; Postaire et al., 2010), Cc by solute accumulation (Munns and Tester, 2008), m and Y by the activities of wall enzymes (Cosgrove, 2005) and P by interactions with any one of the preceding values. Both water deficits and excess salinity in the rhizosphere have been shown to induce reductions in cell and whole-plant growth. In the short term (hours), these may be associated with reductions in cell turgor pressure. Stress-induced losses in turgor pressure can be reversed by increases in solute accumulation, that is, osmotic adjustment. However, even after complete turgor recovery in expanding tissues, growth may continue to be limited by ongoing reductions in rates of cell production and expansion. These may in turn be related to reductions in wall extensibility parameters. This chapter presents an eclectic perspective, based largely on research findings from our laboratory on biochemical and biophysical mechanisms involved in growth inhibition by water deficits, as caused by salinity (Section II), drought (Section III) and most recently, by novel plant interactions with aqueous colloids in soil solutions (Section IV; Fig. 1). Much attention in each section is given to the often-overlooked roles of plant cell walls in regulating whole-plant growth inhibition under stressful conditions. Better understanding of such mechanisms could ideally point to ways of improving the ability of crop plants to maintain growth and hence yield stability. For many additional aspects of plant responses to salt and water stress, the reader is directed to the other chapters of this volume and to many fine review articles that have appeared in recent years, for example, Munns and Tester (2008), Neumann (2008), Walter et al. (2009), Mittler and Blumwald (2010), Skirycx and Inze (2010), Urano et al. (2010) and Yang et al., (2010).
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Sub- or supra-optimal environmental inputs
Sensing mechanisms
Local and long distance signals
Signal transduction
Genomic and post-genomic responses
Alterations in cytoplasmic and apoplastic metabolism
Altered growth and development at level of cell, organ and organism
Acclimation or stress-induced death
Fig. 1. A holistic view of plant interactions with stressful environmental conditions.
II. PLANT GROWTH AND SURVIVAL DURING MODERATE AND SEVERE SALINITY STRESS Excess salinity in the soil solution to which plant roots are exposed can be derived from geochemical sources, sea water infiltration of coastal ground waters, sea water salts in wind and rain, excess fertilizer application to soils or supplementary irrigation with salt-containing irrigation waters (the water evaporates leaving salts to accumulate in the soil). Excessive accumulation of salts in the rhizosphere can lead to growth inhibition, leaf necrosis, accelerated onset of senescence, wilting and death. Different physiological mechanisms can be involved. An osmotic mechanism involves the build-up of salts in the rhizosphere or in the small volume of fluids in the apoplastic cell
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wall compartments. This leads to more negative water potentials which can decrease or even reverse the inwardly directed water potential gradients responsible for water uptake, turgor maintenance and cell expansion (see Eq. (1)). Decreases in water uptake can be gradually reversed, in the case of cells by cytoplasmic accumulation of additional solutes through the process of osmotic adjustment (Evlagon et al., 1990; Munns and Tester, 2008; Neumann et al., 1988) or, in the case of root water uptake, by the development of more negative xylem water potentials. However, growth of maize seedling roots and leaves (but not of bean or rice leaves cf. Lu and Neumann, 1999; Neumann et al., 1988), may continue to be reduced, by parallel salinityinduced reductions in the physical extensibility of the expanding cell walls, even after full osmotic adjustment (cf. Neumann, 1993; Neumann et al., 1994). Interestingly, reduced cell wall extensibility (sometimes termed ‘wall stiffening’) induced by salinity in growing maize leaves can also be induced by exposure of maize seedling roots to osmotic stress alone; thus, toxic effects of sodium or other ions are not necessarily involved. Instead, the regulatory involvement of root to leaf hydraulic signals in initiating decreases in wall extensibility of growing tissues (and stomatal closure) has been postulated (e. g., Chazen and Neumann, 1994; Chazen et al., 1995; Christmann et al., 2007). Excessive salinity in the rhizosphere can also cause hydraulic limitations to leaf growth by inducing regulated decreases in root hydraulic conductivity (Azaizeh and Steudle, 1991; Chazen et al., 1995; Evlagon et al., 1990; Lu and Neumann, 1999). Thus, ongoing research into genomic and epigenetic changes that regulate either cell wall mechanics or plant hydraulics may provide useful approaches for potentially limiting the plant growth inhibition that occurs during water and salinity stress. Another mechanism involved in adverse plant-responses to salinity is associated with excessive, concentration-dependent uptake of salts into affected cells. This can lead to toxic symptoms associated with ionic and hormonal imbalances (Munns and Tester, 2008). The toxicity associated with excess salt accumulation in the cytoplasm has also been mechanistically related to increased generation of reactive oxygen species (ROS). Increased levels of ROS, aside of potential signaling roles, can have damaging effects on essential cellular components such as membranes, proteins and nucleic acids (see reviews by Halliwell and Gutteridge, 1989; Miller et al., 2010). Endogenous or salt-induced increases in levels of antioxidant enzymes and their associated substrates may more or less successfully mitigate the potentially adverse effects of excessive ROS accumulation. For example, a clear correlation was revealed between relatively high endogenous levels of antioxidant activity (enzymes and substrates) in the leaves and roots of a saltresistant wild tomato (Lycopersicon penellii) and lower levels in a much less
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salt-resistant cultivated tomato (Shalata and Tal, 1998; Shalata et al., 2001). Shalata and Neumann (2001) showed that an exogenous supply of ascorbic acid, a water soluble antioxidant substrate found in plants and also known as vitamin C in humans, could mitigate increases in lipid peroxidation caused by highly stressful root exposures of whole, transpiring, tomato seedlings to 300 mM NaCl for up to 9 h. The 9-h salt treatment rapidly and uniformly induced a complete wilting of the shoots, and in the absence of supplementary ascorbic acid, the 9-h treatment was 100% lethal. However, supplementary supplies of ascorbic acid via the roots facilitated a remarkable recovery from wilting and a continuation of apparently normal growth in circa 50% of the wilted seedlings following a return to non-saline root media. This remedial effect was not obtained when roots were supplied equivalent concentrations of other small organic molecules without equivalent antioxidant activity. Subsequently, several studies have provided additional evidence for a potentially protective role of increased levels of either endogenous or exogenous ascorbic acid, in the apoplast as well as the cytoplasm of salinised plants (cf. Athar et al., 2008; Hemavathi et al., 2009; Huang et al., 2005; Yamamoto et al., 2005). However, the practical utility of chemical treatments with exogenous antioxidants, or of using antioxidant traits (among others) in breeding for salinity tolerance, remains to be demonstrated under variable field conditions. Most attempts at improving the resistance of crop plants to salinity stress have rightly focused in recent years on genomic approaches aimed at discovering and utilising genes associated with two vital physiological parameters, that is, salt exclusion and growth maintenance (see reviews by Munns, 1993; Neumann, 1997). The introduction into agricultural practice of genetically engineered crop varieties can, however, be a very prolonged and difficult process even when compared with the time required to introduce new varieties produced by more traditional breeding approaches (cf. Potrykus, 2010; Yang et al., 2010). Fortunately, conventional breeding for enhanced ability to maintain growth and exclude sodium ions can now be accelerated by use of genetic marker technology and appears to be resulting in more saltresistant varieties of major crop species such as maize, rice and wheat (Schubert et al., 2009; personal communications by Rana Munns, CSIRO, Australia and Glen Gregorio, IRRI, Philippines). The process of breeding more resistant varieties may be further accelerated by the recent introduction of industrialised processes for mass characterisation and selection of individuals showing desirable phenotypic traits resulting from phenotypic plasticity in populations of isogenic lines subjected to different environmental challenges (Munns and Tester, 2008; Nicotra and Davidson, 2010). Moreover, the realisation that inheritable epigenetic changes, as well as genomic
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changes are involved in evolutionary selection processes may have important new implications for plant breeding. For example, Hauben et al. (2009) give impressive examples of the potential benefits associated with the exploitation of apparently stable epigenetic variability in breeding programs. They showed that respiratory energy-use efficiency is an important epigenetically regulated factor in determining seed yield in canola (Brassica napus). Thus, individual plants in an isogenic canola population and their selffertilised progenies were recursively selected for respiration intensity and populations with distinct physiological and agronomical characteristics, including increased stress resistance, were isolated. Most importantly, this apparently simple approach appeared to facilitate further improvements in the already high yield potentials of existing commercial hybrids.
III. PLANT GROWTH DURING MODERATE WATER STRESS EPISODES Plant water stress symptoms appear during periods of water deficit when the rate of supply of water from soil to plant falls below the water demands of the combined processes of growth and transpiration. Water deficits can result from drought-induced soil drying, salt-induced osmotic limitations to water uptake, limitations to soil or plant water transport and hot, dry, atmospheric conditions which increase evaporative demands. Drought may take the form of terminal drought that leads progressively to desiccation and death or intermittent drought, such as that occurring between rainfall or supplementary irrigation events. The initial response of whole plants to water deficits involves rapid reductions in leaf (and to a lesser extent root) growth rates followed, sooner or later, by partial or complete closure of stomata. These responses can reduce transpirational water loss at the cost of associated reductions in photosynthetic potential. Moreover, stress-induced root growth inhibition will necessarily limit the capacity of the root system to search for new, untapped water reserves in the soil profile. A practical implication is that breeding or engineering plants for avoidance of growth inhibition during intermittent periods of moderate water stress, or during continued exposure to the osmotic effects of moderate salinity, might offer opportunities for growth and yield stabilisation. Such a goal may benefit from improved understanding of biochemical and genetic mechanisms involved in stress-induced growth inhibition in both roots and leaves. As mentioned in the introductory section, plant growth inhibition can result from hydraulic and/or biomechanical (cell wall) limitations to cell and organ extensibility. This section concerns some recent advances in the
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understanding of mechanisms involved in the biomechanical regulation of growth inhibition during water stress. Bogoslavsky and Neumann (1998) described laboratory studies investigating the relative roles of increased water availability and of apoplastic pH in regulating the recovery of leaf growth from water stress. The responses of the emerging first leaf of whole-maize seedlings with a single primary root were studied. A moderate water deficit, with attendant leaf growth reductions, was first imposed by exposing the roots for 1 h to aerated nutrient solution containing the non-penetrating osmolyte PEG 6000 at a water potential of 0.4 MPa. A micro-syringe fitted with a fine needle was then used to inject 12-mL aliquots of various aqueous solutions into the circa 1-cm long elongation zone at the base of the emerging first leaf of the water-stressed seedlings. Changes in leaf position were determined at 1-s intervals by using electronic position transducers (LVDTs) connected via thread to the leaf tips. The position data was plotted against time and used to simultaneously track instantaneous growth rates in up to 16 individual seedlings. By measuring the effects of gently applying and removing a small force (2 g weight), relative in vivo or in vitro measures of the irreversible component of leaf mechanical extensibility could also be obtained. Injection of water alone into the leaf elongation zone of water-stressed seedlings immediately induced remarkable increases in the (inhibited) rates of leaf elongation. The accelerated growth rate declined gradually to the original low growth rate over about 30 min. Thus, the increased availability of the injected water at 0 MPa appeared to allow increased rates of water uptake for leaf cell expansion. Similar findings were obtained when strong buffer solutions at pH 4.5 were injected in place of water. However, injection of isoosmotic solutions of the same buffers at pH 5.5 largely prevented utilisation of the injected water for growth stimulation. Thus, small upwards changes in the pH of apoplastic solution injected into the elongation zone effectively limited the acceleration of leaf growth normally elicited by local increases in water availability. Injected solutions buffered at pH 5.5 also inhibited the increases in relative leaf tissue extensibility (both in vivo and in vitro) that were induced by injections of water alone or of pH 4.5 buffer. In addition, the acceleration of leaf growth by injected water was also prevented when sodium vanadate or erythrosin B, both inhibitors of proton-pumping ATPases, was included in the injected water. Overall, the findings suggested that the pH 5.5 buffer limited ongoing wall acidification to lower pH values and the associated increases in wall extensibility needed to allow utilisation of the injected water for cell expansion. These findings were consistent with the ‘acid growth hypothesis’, that is, that small changes in apoplastic pH, regulated by outward proton-pumping ATPases in the plasma membranes, can
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affect cell wall proteins such as expansins, XET (xyloglucan endotransglycosylase) and cell wall polysaccharide linkages, thereby effecting wall loosening and increased rates of cell expansion (Cosgrove, 2005; Fry, 1986; Hager et al., 1971; Rayle and Cleland, 1970, 1992). Zo¨rb et al. (2005) and Pitann et al. (2009) provided further evidence for the involvement of protonpumping ATPases in leaf growth inhibition by the osmotic component of salinity stress. Finally, Gevaudant et al. (2007) genetically engineered increased expression in tobacco plants of either a wild-type Hþ ATPase or of a modified Hþ ATPase in which the autoinhibitory domain was removed in order to facilitate constitutive expression. The latter transformant showed aberrant growth but also revealed increases in resistance to growth inhibition by salt stress. Further support for the involvement of the Hþ ATPase and wall acidification in the regulation of cell wall extensibility and growth was provided in several reports in which the more accessible elongation zone at the apex of the maize primary root was investigated (Bassani et al., 2004; Fan and Neumann, 2004; Fan et al., 2006; Zhu et al., 2007). Bassani et al. (2004) used a suppressive subtractive hybridisation technique to reveal that 150 growth-related genes were preferentially expressed in the elongating region of the maize root tip as compared with the subtending fully elongated region. One immediate conclusion is that growth regulation is likely to be a complex, multigenic process (cf. Birnbaum et al., 2003). Interestingly, growth in the accelerating growth region, that is, in the first 3 mm behind the maize primary root tip, is maintained, even under water stress. Bassani et al. used Northern blots to show that transcripts of two candidate genes related to wall metabolism (Hþ ATPase and XET, cf. Wu and Cosgrove, 2000) were highly expressed in this region under both control and water deficit conditions and that transcript expression was relatively down regulated in the more basal decelerating and fully elongated regions. These findings suggested a close spatial association between maintenance of accelerating growth and the expression of these particular genes, as well as several others. Fan and Neumann (2004) more directly investigated the possibility that root growth inhibition during water stress might be related to stress-induced decreases in the ability of proton-pumping ATPases to acidify the expanding cell walls and thereby maintain cell wall extensibility. Again using primary roots of whole-maize seedlings, they revealed clear spatial correlations between the growth-inhibitory effects of a PEG-induced water deficit on segmental elongation rates in the more basal regions of the elongation zone and profiles of proton flux from the root epidermis or of apoplastic pH in epidermal cell walls. They also showed that exogenous acidification of roots
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with succinate buffer at pH 4.5 could partially reverse the stress-induced inhibition of growth. Thus, the more apical zone of accelerating growth responded to acidification with further increases in growth rates while the mid- and basal region of the elongation zone showed limited or zero responses, respectively. This suggested that regulated changes in wall pH were not the only factor involved in regulating root growth inhibition by water deficits and that developmental variations in the responsiveness of the cell walls to pH change were also involved. Fan et al. (2006) provided support for this suggestion. They directly measured the in vitro extensibility of tissues in the three regions of the elongation zone of maize primary roots and confirmed that water deficits did not reduce mechanical extensibility in the region 0–3 mm behind the tip but did do so in more basal regions (cf. Wu and Cosgrove, 2000). More importantly, they used UV fluorescence, Fourier transform IR spectroscopy and a specific stain for lignin to show that an accelerated stelar accumulation of wall-based phenolic compounds and lignin accompanied stress-induced decreases in the extensibility of the basal regions. Moreover, the expression of two gene transcripts involved in lignin synthesis (cinnamyl-CoA reductases 1 and 2) increased within 1 h of initiating water deficits. Water-stressinduced increases in accumulation of UV-fluorescent phenolics in expanding cell walls of soybean roots were also reported by Yamaguchi et al. (2010) suggesting that similar stress-induced processes of cell wall stiffening, by acceleration of phenolic cross linking and lignification, may have evolved in both mono and dicot roots. An obvious question is ‘why have plants evolved and retained a response to water deficits which limits root growth when it is most needed to facilitate the search for additional soil water?’ One possibility suggested by Fan et al. (2006) is that such root growth limitations may increase the relative availability of internal supplies of essential water and solutes to meristematic regions, thereby increasing their chances of surviving drying soil environments and of subsequent root growth recovery, if or when soil water availability is increased by rainfall or irrigation. In conclusion, root and leaf growth regulation by salinity or water stress can involve multiple genetic, biochemical and physiological responses which may help acclimation to the mixed stresses faced by plants in rapidly changing real world environments. Further plant research at all levels of investigation, should facilitate educated trials involving epigenetic trait selection, conventional marker assisted breeding and genetic engineering approaches. These could lead to the introduction of new varieties tailored to specific locales and specific types of stress.
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IV. WHOLE-PLANT WATER AVAILABILITY AND GROWTH CAN ALSO BE LIMITED BY COLLOID STRESS The possibility that the availability of water to higher plants may also be limited by interactions with environmental colloids is generally overlooked. This section briefly introduces recent laboratory findings which indicated the existence of ‘plant colloid stress’. Asli and Neumann (2009) investigated the possibility that colloidal suspensions of inorganic nano-particulate materials of industrial or natural origin, that can be present in soil solutions, could interfere with rhizosphere water transport. They found that external colloidal suspensions of either naturally derived bentonite clay particles or industrially produced TiO2 nanoparticles (30 nm diameter) at 1 gL 1 could significantly reduce water transport through the intact epidermal surfaces of maize primary roots by up to 40%. Moreover, similar additions to the hydroponic solution surrounding the roots of whole-maize seedlings rapidly inhibited leaf growth and transpiration. A subsequent report showed that colloidal suspensions of humic acid at 1 gL 1 had similar inhibitory effects (Asli and Neumann, 2010). Humic acid is an organic colloid that is ubiquitous in soils and soil solutions and may act as a growth stimulant at low concentrations. In addition to reducing root hydraulic conductivity, excessive soil accumulation of humic acid caused an inhibition of shoot growth and reduced plant ability to withstand soil drying. Experiments with model polymers and estimates of cell wall pore sizes suggested that nanosized inorganic or organic particles in soil waters can be transported by mass flow to the root cell wall surfaces of transpiring plants where water flow is reduced by the formation of cake layers and /or by pore blocking. Importantly, the levels of colloids in agricultural soil waters may be further increased when solid wastes and recycled waste waters rich in organic matter are applied to the soil. Recently, the potential for disruption of xylem water transport by physical interactions between endogenous protein colloids in the xylem sap and the cell wall material comprising xylem pit membranes was also reported (Neumann et al., 2010). Thus, ‘colloid stress’ resulting from the inhibitory effects on water transport of physical interactions between plant cell walls and environmental or internal colloids, is a novel stress-factor that may affect plant water relations and further limit plant ability to resist salinity and drought.
REFERENCES Asli, S. and Neumann, P. M. (2009). Colloidal suspensions of clay or titanium dioxide nanoparticles can inhibit leaf growth and transpiration via physical effects on root water transport. Plant, Cell and Environment 32, 577–584.
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Asli, S. and Neumann, P. M. (2010). Rhizosphere humic acid interacts with root cell walls to reduce hydraulic conductivity and plant development. Plant and Soil 336, 313–322. Athar, H. R., Khan, A. and Ashraf, M. (2008). Exogenously applied ascorbic acid alleviates salt-induced oxidative stress in wheat. Environmental and Experimental Botany 63, 224–231. Azaizeh, H. and Steudle, E. (1991). Effects of salinity on water transport of excised maize (Zea mays L.) roots. Plant Physiology 99, 1136–1145. Bassani, M., Neumann, P. M. and Gepstein, S. (2004). Differential expression profiles of growth related genes in the elongation zone of maize primary roots. Plant Molecular Biology 56, 367–380. Birnbaum, K., Shasha, D. E., Wang, J. Y., Jung, J. W., Lambert, G. M., Galbraith, D. W. and Benfey, P. N. (2003). A gene expression map of the Arabidopsis root. Science 302, 1956–1960. Bogoslavsky, L. and Neumann, P. M. (1998). Rapid regulation by acid-pH of cellwall adjustment and leaf growth, in intact maize plants responding to reversal of water stress. Plant Physiology 118, 701–709. Chazen, O. and Neumann, P. M. (1994). Hydraulic signals from the roots and rapid cell wall hardening in growing maize leaves, are primary responses to PEG induced water deficits. Plant Physiology 104, 1385–1392. Chazen, O., Hartung, W. and Neumann, P. M. (1995). The different effects of PEG 6000 and NaCl on leaf development are associated with differential inhibition of root water transport. Plant, Cell and Environment 18, 727–735. Christmann, A., Weiler, E. W., Steudle, E. and Grill, E. (2007). A hydraulic signal in root-to-shoot signalling of water shortage. The Plant Journal 52, 167–174. Cosgrove, D. J. (2005). Growth of the plant cell wall. Nature Reviews Molecular Cell Biology 6, 850–861. Evlagon, D., Ravina, I. and Neumann, P. M. (1990). Interactive effects of salinity and calcium on osmotic adjustment, hydraulic conductivity and growth in primary roots of maize seedlings. Israel Journal of Botany 39, 239–247. Fan, L. and Neumann, P. M. (2004). The spatially variable inhibition by water deficit of maize root growth correlates with altered profiles of proton flux and cell wall pH. Plant Physiology 135, 2291–2300. Fan, L., Linker, R., Gepstein, S., Tanimoto, E., Yamamoto, R. and Neumann, P. M. (2006). Progressive inhibition by water deficit of cell wall extensibility and growth along the elongation zone of maize roots is related to increased lignin metabolism and progressive stelar accumulation of wall phenolics. Plant Physiology 140, 603–612. Fry, S. C. (1986). Cross-linking of matrix polymers in the growing cell walls of angiosperms. Annual Review of Plant Physiology 37, 165–186. Gevaudant, F., Duby, G., von Stedingk, E., Zhao, R., Morsomme, P. and Boutry, M. (2007). Expression of a constitutively activated plasma membrane Hþ ATPase alters plant development and increases salt tolerance. Plant Physiology 144, 1763–1776. Hager, A., Menzel, H. and Krauss, A. (1971). Versuche und hypothese zur primarwirkung des auxins beim shtrekungswachstum. Planta 100, 47–75. Halliwell, B. and Gutteridge, J. M. C. (1989). Protection against oxidants in biological systems: The super oxide theory of oxygen toxicity. In Free Radicals in Biology and Medicine, (B. Halliwell and J. M. Gutteridge, eds.), pp. 86–123. Clarendon Press, Oxford, 0-1985-5294-7.
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Hauben, M., Haesendonckx, B., Standaert, E., Van Der Kelen, K., Azmi, A., Akpo, H., Van Breusegem, F., Guisez, Y., Bots, M., Lambert, B., Laga, B. and De Block, M. (2009). Energy use efficiency is characterized by an epigenetic component that can be directed through artificial selection to increase yield. Proceedings of the National Academy of Sciences 106, 20109–20114. Hemavathi, U. C. P., Ko, E. Y., Nookaraju, A., Reddy, A. C., Chun, S. C., Kim, D. H. and Park, S. W. (2009). Over-expression of strawberry D-galacturonic acid reductase in potato leads to accumulation of vitamin C with enhanced abiotic stress tolerance. Plant Science 177, 659–667. Huang, C., He, W., Guo, J., Chang, X., Su, P. and Zhang, L. (2005). Increased sensitivity to salt stress in an ascorbate-deficient Arabidopsis mutant. Journal of Experimental Botany 56, 3041–3049. Lockhart, J. A. (1965). An analysis of irreversible plant cell elongation. Journal of Theoretical Biology 8, 264–275. Lu, Z. and Neumann, P. M. (1998). Water stressed maize, barley and rice seedlings show species specific diversity in mechanisms of leaf growth inhibition. Journal of Experimental Botany 49, 1945–1952. Lu, Z. and Neumann, P. M. (1999). Water stress inhibits hydraulic conductance and leaf growth in rice seedlings but not transport of water via mercury sensitive water channels in the root. Plant Physiology 120, 143–152. McDowell, N., Pockman, W. T. Allen, C. D. et al. (2008). Mechanisms of plant survival and mortality during drought: Why do some plants survive while others succumb to drought? The New Phytologist 178, 719–739. Miller, G., Suzuki, N., Ciftci-Yilmaz, S. and Mittler, R. (2010). Reactive oxygen species homeostasis and signaling during drought and salinity stresses. Plant, Cell & Environment 33, 453–467. Mittler, R. and Blumwald, E. (2010). Genetic engineering for modern agriculture: Challenges and perspectives. Annual Review of Plant Biology 61, 443–462. Munns, R. (1993). Physiological processes limiting growth in saline soils: Some dogmas and hypotheses. Plant, Cell & Environment 16, 15–24. Munns, R. and Tester, M. (2008). Mechanisms of salinity tolerance. Annual Review of Plant Biology 59, 651–681. Neumann, P. M. (1993). Rapid and reversible modifications of extension capacity of cell walls in elongating maize leaf tissues responding to root addition and removal of NaCl. Plant, Cell & Environment 16, 1107–1111. Neumann, P. (1997). Salinity resistance and plant growth revisited. Plant, Cell & Environment 20, 1193–1198. Neumann, P. M. (2008). Coping mechanisms for crop plants in drought prone environments. Annals of Botany 101, 901–907. Neumann, P. M., Van Volkenburgh, E. and Cleland, R. E. (1988). Salinity stress reduces bean leaf expansion and turgor but not cell wall extensibility. Plant Physiology 88, 233–237. Neumann, P. M., Azaizeh, H. and Leon, D. (1994). Hardening of root cell walls: A growth inhibitory response to salinity stress. Plant, Cell and Environment 17, 303–309. Neumann, P. M., Weissman, R., Stefano, G. and Mancuso, S. (2010). Accumulation of xylem transported protein at pit membranes and associated reductions in hydraulic conductance. Journal of Experimental Botany 61, 1711–1717. Nicotra, A. B. and Davidson, A. (2010). Adaptive phenotypic plasticity and plant water use. Functional Plant Biology 37, 117–127. Pitann, B., Schubert, S. and Muhling, K. H. (2009). decline in leaf growth under salt stress is due to an inhibition of Hþ pumping activity and increase in
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apoplastic pH of maize leaves. Journal of Plant Nutrition and Soil Science 172, 535–543. Postaire, O., Tournaire-Roux, C., Grondin, A., Boursiac, Y., Scha¨ffner, A. R., Morillon, R. and Maurel, C. (2010). A PIP1 aquaporin contributes to hydrostatic pressure-induced water transport in both the root and rosette of Arabidopsis. Plant Physiology 152, 1418–1430. Potrykus, I. (2010). Insights from Golden Rice: GE-projects for public good are faced with inhibitive conditions. ASPB annual meeting—Plant Biology 2010, Montreal, Canada, Abs # S042, http://abstracts.aspb.org/pb2010/public/. Rayle, D. L. and Cleland, R. E. (1970). Enhancement of wall loosening and elongation by acid solution. Plant Physiology 46, 250–253. Rayle, D. L. and Cleland, R. E. (1992). The acid growth theory of auxin induced cell elongation is alive and well. Plant Physiology 99, 1271–1274. Schubert, S., Neubert, A., Schierholt, A., Sumer, A. and Zorb, C. (2009). Development of salt-resistant maize hybrids: The combination of physiological strategies using conventional breeding methods. Plant Science 177, 196–202. Shalata, A. and Neumann, P. M. (2001). Exogenous ascorbic acid (vitamin C) increases resistance to salt stress and reduces lipid peroxidation. Journal of Experimental Botany 52, 2207–2211. Shalata, A. and Tal, M. (1998). The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii. Physiologia Plantarum 104, 169–174. Shalata, A., Mittova, V., Volokita, M., Guy, M. and Tal, M. (2001). Response of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii to salt-dependent oxidative stress: The root antioxidative system. Physiologia Plantarum 112, 487–494. Skirycx, A. and Inze, D. (2010). More from less: Plant growth under limited water. Current Opinion in Biotechnology 21, 197–203. Sperry, J. S., Hacke, U. G., Oren, R. and Comstock, J. P. (2002). Water deficits and hydraulic limits to leaf water supply. Plant, Cell and Environment 25, 251–263. Urano, K., Kurihara, Y., Seki, M. and Shinozaki, K. (2010). ‘Omics’ analyses of regulatory networks in plant abiotic stress responses. Current Opinion in Plant Biology 13, 132–138. Uyttewaal, M., Traas, J. and Hamant, O. (2010). Integrating physical stress, growth, and development. Current Opinion in Plant Biology 13, 46–52. Walter, A., Silk, W. K. and Schurr, U. (2009). Environmental effects on spatial and temporal patterns of leaf and root growth. Annual Review of Plant Biology 60, 279–304. Watson, J. D. and Crick, F. H. C. (1953). A structure for deoxyribose nucleic acid. Nature 171, 737–738. Wu, Y. J. and Cosgrove, D. J. (2000). Adaptation of roots to low water potentials by changes in cell wall extensibility and cell wall proteins. Journal of Experimental Botany 51, 1543–1553. Yamaguchi, M., Valliyodan, B., Zhang, J., Lenoble, M. E., Yu, O., Rogers, E. E., Nguyen, H. T. and Sharp, R. E. (2010). Regulation of growth response to water stress in the soybean primary root. I. Proteomic analysis reveals region-specific regulation of phenylpropanoid metabolism and control of free iron in the elongation zone. Plant Cell and Environment 33, 223–243. Yamamoto, A., Bhuiyan, M. N. H., Waditee, R., Tanaoka, Y., Esaka, M., Oba, K., Jagendorf, A. T. and Takabe, T. (2005). Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and arabidopsis plants. Journal of Experimental Botany 56, 1785–1796.
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Yang, S., Vanderbeld, B., Wan, J. and Huang, Y. (2010). Narrowing down the targets: Towards successful genetic engineering of drought-tolerant crops. Molecular Plant 3, 469–490. Zhu, J. M., Alvarez, S., Marsh, E. L., LeNoble, M. E., Cho, I. J., Sivaguru, M., Chen, S. X., Nguyen, H. T., Wu, Y. J., Schachtman, D. P. and Sharp, R. E. (2007). Cell wall proteome in the maize primary root elongation zone. II. Region-specific changes in water soluble and lightly ionically bound proteins under water deficit. Plant Physiology 145, 1533–1548. Zo¨rb, C., Stracke, B., Tramnitz, B., Denter, D., Su¨mer, A., Mu¨hling, K. H., Yan, F. and Schubert, S. (2005). Does Hþ pumping by plasmalemma ATPase limit leaf growth of maize (Zea mays) during the first phase of salt stress? Journal of Plant Nutrition and Soil Science 168, 550–557.
PETER M. NEUMANN1
Department of Environmental, Water and Agricultural Engineering, Technion Israel Institute of Technology, Haifa, Israel
I. II. III. IV.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plant Growth and Survival During Moderate and Severe Salinity Stress . . . Plant Growth During Moderate Water Stress Episodes . . . . . . . . . . . . . . . . . . . . . Whole-Plant Water Availability and Growth Can also be Limited by Colloid Stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT This chapter presents an eclectic perspective, based largely on research findings from our laboratory on biochemical and biophysical mechanisms involved in growth inhibition by water deficits, as caused by salinity (Section II), drought (Section III) and recently reported plant interactions with aqueous colloids found in soil solutions or xylem sap (Section IV). Much attention in each section is given to the roles of plant cell walls in regulating whole-plant growth inhibition under stressful conditions. One conclusion is that ongoing research into genomic and epigenetic changes that participate in the regulation of cell wall changes, cellular antioxidant status and plant hydraulics may provide new approaches for limiting the plant growth inhibition or mortality associated with salinity and drought. In Section IV, it is concluded that the ‘colloid stress’ that results from the inhibitory effects on water transport of physical
1
Corresponding author: E-mail: [email protected]
Advances in Botanical Research, Vol. 57 Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00 DOI: 10.1016/B978-0-12-387692-8.00002-3
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interactions between plant cell walls and environmental or internal colloids is a novel stress-factor that can affect plant water relations and further limit plant ability to resist salinity and drought.
I. INTRODUCTION Abiotic stress can be defined as an adverse situation resulting from plant exposure to sub- or supra-optimal levels of environmental inputs such as water, light, temperature or solutes. Abiotic stress resulting from water deficits or excessive salinity generally leads to reductions in photosynthesis, transpiration and growth. Such stresses are a major, world-wide-factor limiting the ability of mankind to produce adequate supplies of plant food, fibre and fuel for an expanding world population. Ongoing advances in botanical research into plant responses to abiotic stresses, in addition to increasing basic knowledge, may facilitate advances in our practical ability to maintain and increase plant productivity in stressful agricultural or natural environments. Botany, or that branch of biology that concerns the scientific study of plant life, has evolved rapidly since Watson and Crick (1953) published their seminal paper on DNA structure. The genomic era they signalled has been characterised by the introduction of increasingly sophisticated analytical technologies that now facilitate ‘deep’ genomic, transcriptomic, proteomic and metabolomic investigations. Most recently, there has been increasing interest in hereditable epigenetic changes involving DNA methylation, histone-modification and microRNA. These developments in cell molecular biology are leading to a post-genomic era where systems biology seeks to integrate understanding of the processes regulating metabolism into holistic models of development, albeit mainly at the cellular level. Parallel advances in research into transport and communication processes that are associated with development at the level of tissues, organs and whole organisms, are also needed for fuller understanding and more successful modifications of whole-plant responses to environmental change. An important area in which further study is certainly needed is the extracellular (apoplastic) region of plants, that is, plant cell walls, xylem and environmental boundary layers at root and leaf surfaces. Each of these can make direct ‘post-genomic’ contributions to the regulation of whole-plant growth and development under optimal or stressful conditions. An example of apoplastic changes that may regulate plant development is given by what happens as tension, that is, negative hydrostatic pressure, increases in the xylem water columns of plants facing water deficits. When critical tensions are reached, the formation of gaseous embolisms may disrupt xylem hydraulic continuity and thereby restrict essential xylem-transport of soil derived
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water and solutes to the shoot. Such physical reductions in water transport can then adversely affect leaf functioning and hence, plant growth and development (McDowell et al., 2008; Sperry et al., 2002). Much of the emphasis in this chapter is on apoplastic changes that are involved in the regulation of cell, organ and whole-plant growth responses to salinity and water stress. Such stresses can be moderate, that is, non-lethal, or extreme, that is, lethal. A central theme is that the molecular changes induced in plant cells by moderate environmental stresses will usually lead to rapid decrease in rates of growth of tissues and organs, be they roots, stems, leaves, flowers, fruits or seeds. Stress-induced changes in plant growth rates, tissue mechanics and phenology can therefore provide integrated reflections of underlying molecular changes (Hauben et al., 2009; Neumann, 1997; Nicotra and Davidson, 2010; Uyttewaal et al., 2010). The practical consequences of stress-induced decreases in the growth of plants can be viewed in different ways. For example, limiting new leaf area production under water stress not only will limit plant potential for photosynthesis but will also limit overall leaf transpiration. It may therefore extend the availability of limited soil water reserves and hence plant ability to survive long enough to produce (a limited amount of) seeds. Albeit, in an agricultural context and in the viewpoint of this chapter, stress-induced reductions in growth rates of crop plants will generally be associated with unwanted reductions in economic yields. Thus, stress acclimation that involves growth inhibition and associated increases in plant survival may still be viewed as a hindrance by farmers. In order to review the plant growth reductions induced by environmental stresses, it is necessary to first consider what exactly is meant by the term growth. Growth can be defined as an irreversible increase in size. In plants, vegetative and reproductive growth is always based on the expansion of young daughter cells produced by ongoing meristematic divisions. Growth is a prerequisite for plant development, and its regulation may occur via changes in the cell division cycle and in rates or duration of cell expansion (cf. Lu and Neumann, 1998; Skirycx and Inze, 2010). Note, however, that even cell division is dependent on a limited amount of prior cell expansion. The processes involved in the cell expansion that is primarily responsible for size increases at tissue, organ and whole-plant levels can be conveniently described by the Lockhart (1965) model equations. The first equation states that relative rates of cell expansion growth (RGR) are limited by hydraulic factors which control the entry of water required for cell volume increases. One such hydraulic factor is the maintenance of cytoplasmic water potential (Cc) at a level which is more negative than that of the external water source (Co). The resultant water potential gradient provides the driving force for
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water uptake and resultant flow is in turn, modulated by an additional factor, that is, the hydraulic conductivity, L, of the water conducting pathways leading into the cytoplasm. RGR ¼ LðCo Cc Þ
ð1Þ
The second Lockhart equation indicates that relative rates of cell growth are also co-regulated by interactions between cell turgor pressure (P), the mechanical extensibility of the cell walls (m) and the minimum turgor pressure required to start cell expansion, that is, the cell wall yield threshold (Y). RGR ¼ mðP Y Þ
ð2Þ
An often overlooked but important point is that each of the cellular parameters involved (Cc, L, P, m, Y) and hence cell growth itself, can be metabolically regulated. For example, L can be influenced by the opening or closure of aquaporin water channels in the membranes (e.g., Lu and Neumann, 1999; Postaire et al., 2010), Cc by solute accumulation (Munns and Tester, 2008), m and Y by the activities of wall enzymes (Cosgrove, 2005) and P by interactions with any one of the preceding values. Both water deficits and excess salinity in the rhizosphere have been shown to induce reductions in cell and whole-plant growth. In the short term (hours), these may be associated with reductions in cell turgor pressure. Stress-induced losses in turgor pressure can be reversed by increases in solute accumulation, that is, osmotic adjustment. However, even after complete turgor recovery in expanding tissues, growth may continue to be limited by ongoing reductions in rates of cell production and expansion. These may in turn be related to reductions in wall extensibility parameters. This chapter presents an eclectic perspective, based largely on research findings from our laboratory on biochemical and biophysical mechanisms involved in growth inhibition by water deficits, as caused by salinity (Section II), drought (Section III) and most recently, by novel plant interactions with aqueous colloids in soil solutions (Section IV; Fig. 1). Much attention in each section is given to the often-overlooked roles of plant cell walls in regulating whole-plant growth inhibition under stressful conditions. Better understanding of such mechanisms could ideally point to ways of improving the ability of crop plants to maintain growth and hence yield stability. For many additional aspects of plant responses to salt and water stress, the reader is directed to the other chapters of this volume and to many fine review articles that have appeared in recent years, for example, Munns and Tester (2008), Neumann (2008), Walter et al. (2009), Mittler and Blumwald (2010), Skirycx and Inze (2010), Urano et al. (2010) and Yang et al., (2010).
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Sub- or supra-optimal environmental inputs
Sensing mechanisms
Local and long distance signals
Signal transduction
Genomic and post-genomic responses
Alterations in cytoplasmic and apoplastic metabolism
Altered growth and development at level of cell, organ and organism
Acclimation or stress-induced death
Fig. 1. A holistic view of plant interactions with stressful environmental conditions.
II. PLANT GROWTH AND SURVIVAL DURING MODERATE AND SEVERE SALINITY STRESS Excess salinity in the soil solution to which plant roots are exposed can be derived from geochemical sources, sea water infiltration of coastal ground waters, sea water salts in wind and rain, excess fertilizer application to soils or supplementary irrigation with salt-containing irrigation waters (the water evaporates leaving salts to accumulate in the soil). Excessive accumulation of salts in the rhizosphere can lead to growth inhibition, leaf necrosis, accelerated onset of senescence, wilting and death. Different physiological mechanisms can be involved. An osmotic mechanism involves the build-up of salts in the rhizosphere or in the small volume of fluids in the apoplastic cell
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wall compartments. This leads to more negative water potentials which can decrease or even reverse the inwardly directed water potential gradients responsible for water uptake, turgor maintenance and cell expansion (see Eq. (1)). Decreases in water uptake can be gradually reversed, in the case of cells by cytoplasmic accumulation of additional solutes through the process of osmotic adjustment (Evlagon et al., 1990; Munns and Tester, 2008; Neumann et al., 1988) or, in the case of root water uptake, by the development of more negative xylem water potentials. However, growth of maize seedling roots and leaves (but not of bean or rice leaves cf. Lu and Neumann, 1999; Neumann et al., 1988), may continue to be reduced, by parallel salinityinduced reductions in the physical extensibility of the expanding cell walls, even after full osmotic adjustment (cf. Neumann, 1993; Neumann et al., 1994). Interestingly, reduced cell wall extensibility (sometimes termed ‘wall stiffening’) induced by salinity in growing maize leaves can also be induced by exposure of maize seedling roots to osmotic stress alone; thus, toxic effects of sodium or other ions are not necessarily involved. Instead, the regulatory involvement of root to leaf hydraulic signals in initiating decreases in wall extensibility of growing tissues (and stomatal closure) has been postulated (e. g., Chazen and Neumann, 1994; Chazen et al., 1995; Christmann et al., 2007). Excessive salinity in the rhizosphere can also cause hydraulic limitations to leaf growth by inducing regulated decreases in root hydraulic conductivity (Azaizeh and Steudle, 1991; Chazen et al., 1995; Evlagon et al., 1990; Lu and Neumann, 1999). Thus, ongoing research into genomic and epigenetic changes that regulate either cell wall mechanics or plant hydraulics may provide useful approaches for potentially limiting the plant growth inhibition that occurs during water and salinity stress. Another mechanism involved in adverse plant-responses to salinity is associated with excessive, concentration-dependent uptake of salts into affected cells. This can lead to toxic symptoms associated with ionic and hormonal imbalances (Munns and Tester, 2008). The toxicity associated with excess salt accumulation in the cytoplasm has also been mechanistically related to increased generation of reactive oxygen species (ROS). Increased levels of ROS, aside of potential signaling roles, can have damaging effects on essential cellular components such as membranes, proteins and nucleic acids (see reviews by Halliwell and Gutteridge, 1989; Miller et al., 2010). Endogenous or salt-induced increases in levels of antioxidant enzymes and their associated substrates may more or less successfully mitigate the potentially adverse effects of excessive ROS accumulation. For example, a clear correlation was revealed between relatively high endogenous levels of antioxidant activity (enzymes and substrates) in the leaves and roots of a saltresistant wild tomato (Lycopersicon penellii) and lower levels in a much less
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salt-resistant cultivated tomato (Shalata and Tal, 1998; Shalata et al., 2001). Shalata and Neumann (2001) showed that an exogenous supply of ascorbic acid, a water soluble antioxidant substrate found in plants and also known as vitamin C in humans, could mitigate increases in lipid peroxidation caused by highly stressful root exposures of whole, transpiring, tomato seedlings to 300 mM NaCl for up to 9 h. The 9-h salt treatment rapidly and uniformly induced a complete wilting of the shoots, and in the absence of supplementary ascorbic acid, the 9-h treatment was 100% lethal. However, supplementary supplies of ascorbic acid via the roots facilitated a remarkable recovery from wilting and a continuation of apparently normal growth in circa 50% of the wilted seedlings following a return to non-saline root media. This remedial effect was not obtained when roots were supplied equivalent concentrations of other small organic molecules without equivalent antioxidant activity. Subsequently, several studies have provided additional evidence for a potentially protective role of increased levels of either endogenous or exogenous ascorbic acid, in the apoplast as well as the cytoplasm of salinised plants (cf. Athar et al., 2008; Hemavathi et al., 2009; Huang et al., 2005; Yamamoto et al., 2005). However, the practical utility of chemical treatments with exogenous antioxidants, or of using antioxidant traits (among others) in breeding for salinity tolerance, remains to be demonstrated under variable field conditions. Most attempts at improving the resistance of crop plants to salinity stress have rightly focused in recent years on genomic approaches aimed at discovering and utilising genes associated with two vital physiological parameters, that is, salt exclusion and growth maintenance (see reviews by Munns, 1993; Neumann, 1997). The introduction into agricultural practice of genetically engineered crop varieties can, however, be a very prolonged and difficult process even when compared with the time required to introduce new varieties produced by more traditional breeding approaches (cf. Potrykus, 2010; Yang et al., 2010). Fortunately, conventional breeding for enhanced ability to maintain growth and exclude sodium ions can now be accelerated by use of genetic marker technology and appears to be resulting in more saltresistant varieties of major crop species such as maize, rice and wheat (Schubert et al., 2009; personal communications by Rana Munns, CSIRO, Australia and Glen Gregorio, IRRI, Philippines). The process of breeding more resistant varieties may be further accelerated by the recent introduction of industrialised processes for mass characterisation and selection of individuals showing desirable phenotypic traits resulting from phenotypic plasticity in populations of isogenic lines subjected to different environmental challenges (Munns and Tester, 2008; Nicotra and Davidson, 2010). Moreover, the realisation that inheritable epigenetic changes, as well as genomic
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changes are involved in evolutionary selection processes may have important new implications for plant breeding. For example, Hauben et al. (2009) give impressive examples of the potential benefits associated with the exploitation of apparently stable epigenetic variability in breeding programs. They showed that respiratory energy-use efficiency is an important epigenetically regulated factor in determining seed yield in canola (Brassica napus). Thus, individual plants in an isogenic canola population and their selffertilised progenies were recursively selected for respiration intensity and populations with distinct physiological and agronomical characteristics, including increased stress resistance, were isolated. Most importantly, this apparently simple approach appeared to facilitate further improvements in the already high yield potentials of existing commercial hybrids.
III. PLANT GROWTH DURING MODERATE WATER STRESS EPISODES Plant water stress symptoms appear during periods of water deficit when the rate of supply of water from soil to plant falls below the water demands of the combined processes of growth and transpiration. Water deficits can result from drought-induced soil drying, salt-induced osmotic limitations to water uptake, limitations to soil or plant water transport and hot, dry, atmospheric conditions which increase evaporative demands. Drought may take the form of terminal drought that leads progressively to desiccation and death or intermittent drought, such as that occurring between rainfall or supplementary irrigation events. The initial response of whole plants to water deficits involves rapid reductions in leaf (and to a lesser extent root) growth rates followed, sooner or later, by partial or complete closure of stomata. These responses can reduce transpirational water loss at the cost of associated reductions in photosynthetic potential. Moreover, stress-induced root growth inhibition will necessarily limit the capacity of the root system to search for new, untapped water reserves in the soil profile. A practical implication is that breeding or engineering plants for avoidance of growth inhibition during intermittent periods of moderate water stress, or during continued exposure to the osmotic effects of moderate salinity, might offer opportunities for growth and yield stabilisation. Such a goal may benefit from improved understanding of biochemical and genetic mechanisms involved in stress-induced growth inhibition in both roots and leaves. As mentioned in the introductory section, plant growth inhibition can result from hydraulic and/or biomechanical (cell wall) limitations to cell and organ extensibility. This section concerns some recent advances in the
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understanding of mechanisms involved in the biomechanical regulation of growth inhibition during water stress. Bogoslavsky and Neumann (1998) described laboratory studies investigating the relative roles of increased water availability and of apoplastic pH in regulating the recovery of leaf growth from water stress. The responses of the emerging first leaf of whole-maize seedlings with a single primary root were studied. A moderate water deficit, with attendant leaf growth reductions, was first imposed by exposing the roots for 1 h to aerated nutrient solution containing the non-penetrating osmolyte PEG 6000 at a water potential of 0.4 MPa. A micro-syringe fitted with a fine needle was then used to inject 12-mL aliquots of various aqueous solutions into the circa 1-cm long elongation zone at the base of the emerging first leaf of the water-stressed seedlings. Changes in leaf position were determined at 1-s intervals by using electronic position transducers (LVDTs) connected via thread to the leaf tips. The position data was plotted against time and used to simultaneously track instantaneous growth rates in up to 16 individual seedlings. By measuring the effects of gently applying and removing a small force (2 g weight), relative in vivo or in vitro measures of the irreversible component of leaf mechanical extensibility could also be obtained. Injection of water alone into the leaf elongation zone of water-stressed seedlings immediately induced remarkable increases in the (inhibited) rates of leaf elongation. The accelerated growth rate declined gradually to the original low growth rate over about 30 min. Thus, the increased availability of the injected water at 0 MPa appeared to allow increased rates of water uptake for leaf cell expansion. Similar findings were obtained when strong buffer solutions at pH 4.5 were injected in place of water. However, injection of isoosmotic solutions of the same buffers at pH 5.5 largely prevented utilisation of the injected water for growth stimulation. Thus, small upwards changes in the pH of apoplastic solution injected into the elongation zone effectively limited the acceleration of leaf growth normally elicited by local increases in water availability. Injected solutions buffered at pH 5.5 also inhibited the increases in relative leaf tissue extensibility (both in vivo and in vitro) that were induced by injections of water alone or of pH 4.5 buffer. In addition, the acceleration of leaf growth by injected water was also prevented when sodium vanadate or erythrosin B, both inhibitors of proton-pumping ATPases, was included in the injected water. Overall, the findings suggested that the pH 5.5 buffer limited ongoing wall acidification to lower pH values and the associated increases in wall extensibility needed to allow utilisation of the injected water for cell expansion. These findings were consistent with the ‘acid growth hypothesis’, that is, that small changes in apoplastic pH, regulated by outward proton-pumping ATPases in the plasma membranes, can
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affect cell wall proteins such as expansins, XET (xyloglucan endotransglycosylase) and cell wall polysaccharide linkages, thereby effecting wall loosening and increased rates of cell expansion (Cosgrove, 2005; Fry, 1986; Hager et al., 1971; Rayle and Cleland, 1970, 1992). Zo¨rb et al. (2005) and Pitann et al. (2009) provided further evidence for the involvement of protonpumping ATPases in leaf growth inhibition by the osmotic component of salinity stress. Finally, Gevaudant et al. (2007) genetically engineered increased expression in tobacco plants of either a wild-type Hþ ATPase or of a modified Hþ ATPase in which the autoinhibitory domain was removed in order to facilitate constitutive expression. The latter transformant showed aberrant growth but also revealed increases in resistance to growth inhibition by salt stress. Further support for the involvement of the Hþ ATPase and wall acidification in the regulation of cell wall extensibility and growth was provided in several reports in which the more accessible elongation zone at the apex of the maize primary root was investigated (Bassani et al., 2004; Fan and Neumann, 2004; Fan et al., 2006; Zhu et al., 2007). Bassani et al. (2004) used a suppressive subtractive hybridisation technique to reveal that 150 growth-related genes were preferentially expressed in the elongating region of the maize root tip as compared with the subtending fully elongated region. One immediate conclusion is that growth regulation is likely to be a complex, multigenic process (cf. Birnbaum et al., 2003). Interestingly, growth in the accelerating growth region, that is, in the first 3 mm behind the maize primary root tip, is maintained, even under water stress. Bassani et al. used Northern blots to show that transcripts of two candidate genes related to wall metabolism (Hþ ATPase and XET, cf. Wu and Cosgrove, 2000) were highly expressed in this region under both control and water deficit conditions and that transcript expression was relatively down regulated in the more basal decelerating and fully elongated regions. These findings suggested a close spatial association between maintenance of accelerating growth and the expression of these particular genes, as well as several others. Fan and Neumann (2004) more directly investigated the possibility that root growth inhibition during water stress might be related to stress-induced decreases in the ability of proton-pumping ATPases to acidify the expanding cell walls and thereby maintain cell wall extensibility. Again using primary roots of whole-maize seedlings, they revealed clear spatial correlations between the growth-inhibitory effects of a PEG-induced water deficit on segmental elongation rates in the more basal regions of the elongation zone and profiles of proton flux from the root epidermis or of apoplastic pH in epidermal cell walls. They also showed that exogenous acidification of roots
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with succinate buffer at pH 4.5 could partially reverse the stress-induced inhibition of growth. Thus, the more apical zone of accelerating growth responded to acidification with further increases in growth rates while the mid- and basal region of the elongation zone showed limited or zero responses, respectively. This suggested that regulated changes in wall pH were not the only factor involved in regulating root growth inhibition by water deficits and that developmental variations in the responsiveness of the cell walls to pH change were also involved. Fan et al. (2006) provided support for this suggestion. They directly measured the in vitro extensibility of tissues in the three regions of the elongation zone of maize primary roots and confirmed that water deficits did not reduce mechanical extensibility in the region 0–3 mm behind the tip but did do so in more basal regions (cf. Wu and Cosgrove, 2000). More importantly, they used UV fluorescence, Fourier transform IR spectroscopy and a specific stain for lignin to show that an accelerated stelar accumulation of wall-based phenolic compounds and lignin accompanied stress-induced decreases in the extensibility of the basal regions. Moreover, the expression of two gene transcripts involved in lignin synthesis (cinnamyl-CoA reductases 1 and 2) increased within 1 h of initiating water deficits. Water-stressinduced increases in accumulation of UV-fluorescent phenolics in expanding cell walls of soybean roots were also reported by Yamaguchi et al. (2010) suggesting that similar stress-induced processes of cell wall stiffening, by acceleration of phenolic cross linking and lignification, may have evolved in both mono and dicot roots. An obvious question is ‘why have plants evolved and retained a response to water deficits which limits root growth when it is most needed to facilitate the search for additional soil water?’ One possibility suggested by Fan et al. (2006) is that such root growth limitations may increase the relative availability of internal supplies of essential water and solutes to meristematic regions, thereby increasing their chances of surviving drying soil environments and of subsequent root growth recovery, if or when soil water availability is increased by rainfall or irrigation. In conclusion, root and leaf growth regulation by salinity or water stress can involve multiple genetic, biochemical and physiological responses which may help acclimation to the mixed stresses faced by plants in rapidly changing real world environments. Further plant research at all levels of investigation, should facilitate educated trials involving epigenetic trait selection, conventional marker assisted breeding and genetic engineering approaches. These could lead to the introduction of new varieties tailored to specific locales and specific types of stress.
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IV. WHOLE-PLANT WATER AVAILABILITY AND GROWTH CAN ALSO BE LIMITED BY COLLOID STRESS The possibility that the availability of water to higher plants may also be limited by interactions with environmental colloids is generally overlooked. This section briefly introduces recent laboratory findings which indicated the existence of ‘plant colloid stress’. Asli and Neumann (2009) investigated the possibility that colloidal suspensions of inorganic nano-particulate materials of industrial or natural origin, that can be present in soil solutions, could interfere with rhizosphere water transport. They found that external colloidal suspensions of either naturally derived bentonite clay particles or industrially produced TiO2 nanoparticles (30 nm diameter) at 1 gL 1 could significantly reduce water transport through the intact epidermal surfaces of maize primary roots by up to 40%. Moreover, similar additions to the hydroponic solution surrounding the roots of whole-maize seedlings rapidly inhibited leaf growth and transpiration. A subsequent report showed that colloidal suspensions of humic acid at 1 gL 1 had similar inhibitory effects (Asli and Neumann, 2010). Humic acid is an organic colloid that is ubiquitous in soils and soil solutions and may act as a growth stimulant at low concentrations. In addition to reducing root hydraulic conductivity, excessive soil accumulation of humic acid caused an inhibition of shoot growth and reduced plant ability to withstand soil drying. Experiments with model polymers and estimates of cell wall pore sizes suggested that nanosized inorganic or organic particles in soil waters can be transported by mass flow to the root cell wall surfaces of transpiring plants where water flow is reduced by the formation of cake layers and /or by pore blocking. Importantly, the levels of colloids in agricultural soil waters may be further increased when solid wastes and recycled waste waters rich in organic matter are applied to the soil. Recently, the potential for disruption of xylem water transport by physical interactions between endogenous protein colloids in the xylem sap and the cell wall material comprising xylem pit membranes was also reported (Neumann et al., 2010). Thus, ‘colloid stress’ resulting from the inhibitory effects on water transport of physical interactions between plant cell walls and environmental or internal colloids, is a novel stress-factor that may affect plant water relations and further limit plant ability to resist salinity and drought.
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