PHYSIOLOGICAL AND MORPHOLOGICAL RESPONSES OF PERENNIAL FORAGES TO STRESS Matt A. Sanderson', David W. Stair2,and Mark A. Hussey2 'Texas A&M University Agricultural Research and Extension Center Stephenville, Texas 76401 *Department of Soil and Crop Sciences Texas A&M University College Station, Texas 77843
I. Introduction 11. Water Deficit A. Whole-Plant Responses B. Effects on Photosynthesis C. Tall Fescue and Endophyte D. Water-Deficit Effects on Forage Quality E. Seedling Establishment in Grasses III. Defoliation Stress A. Whole-Plant Responses B. Remobilization of Carbon and Nitrogen IV LowLight A. Shade Responses B. Light Quality V. Nutrient Stress A. Nitrogen B. Phosphorus C. Acid Soils and Al Toxicity D. Calcareous Soils and Fe-Deficiency Chlorosis VI. Low-Temperature Stress A. Chilling Stress B. Acclimation to Low Temperature and Development of Freezing Tolerance C. Low-Temperature Stress and Organic Reserves D. Tissue Culture and Gene Expression in Low-Temperature Stress VII. Salt Stress A. Salt Accumulation B. Seedling and Adult Plant Responses C. Tissue Culture and Gene Expression in Salt Stress 171 Advanres m Agmnmny, Volume 59 Copyright 0 1997 by Academic Press, Inc. All rights of reproduction in any form reserved.
M. A. SANDERSON ET AL. VIII. Plant Breeding for Abiotic Stress Tolerance A. Defoliation Tolerance B. Drought Tolerance C. Nutrient Stress D. Salinity Tolerance References
I. INTRODUCTION Stress reduces crop growth on nearly all arable land on earth (Solh, 1993) and severely limits agricultural productivity (Boyer, 1982). Perennial forages are grown in many different environments and must endure stresses not normally encountered by annual crops such as repeated defoliation by machines and surviving seasonal extremes in climatic conditions during several years. Forage crops account for 60-90% of feedstuff input for animal production systems (Barnes and Baylor, 1994). Rarely is the abiotic or biotic environment optimum for growth of perennial forages. Indeed, stress may be a regular feature of a particular environment. Stress has been defined as “any factor that decreases plant growth and reproduction below the genotype’s potential” (Osmond et al., 1987).Abiotic stresses include water deficit, temperatureextremes, nutrient imbalances or deficiencies, light extremes, and soil factors (e.g., salinity and pH). Perennial forages commonly are grown on soils of low water-holding capacity or infrequent irrigation, limited fertility, or high salt content. Furthermore, forages must endure subzero temperatures during winter or, in the case of tropical and subtropical forages, withstand chilling or infrequent frosts, and periodic defoliation (e.g., machine harvest). Collectively, stresses may reduce the harvested forage yield, alter its nutritive value, and change species composition of the sward. With the current societal emphasis on sustainable agricultural systems, forage crop production will become more important and, thus, our knowledge of how abiotic stresses limit forage production must increase. There are several reviews on various aspects of the general topic of abiotic stress in plants, particularly in grain crops and plants grown in extensive systems (Jones et al., 1989; Alscher and Cummings, 1990; Fowden et al., 1993; Bohnert er al., 1995). We chose to limit the scope of this review to perennial forages because of limited coverage in previous reviews. We focus on defoliation (primarily machine harvest, not herbivory), low-temperature, water-deficit, nutrient, and salinity stresses and discuss the manifestations of these stresses at the whole-plant and organ level, examine the cellular bases of stress reactions, and explore the genetics of abiotic stress in relation to plant breeding for development of more stressresistant germ plasm. We have attempted to highlight the most recent research and
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refer the reader to the pertinent, in-depth reviews in each of the appropriate sections.
II. WATER DEFICIT A. WHOLE-PLANTRESPONSES For a plant to assimilate atmospheric CO,, it must encounter evapotranspirational water loss and some level of water stress. Some forages escape water stress by completing their life cycle before water becomes limiting [e.g, crested wheatgrass (Agropryron cristutum (L.) Beauv. ssp. pectinarum (Bieb.) Tzvel.) (Bittman and Simpson, 1989; Frank, 1994)l; however, most perennial forages resist water stress with a combination of avoidance and tolerance mechanisms. Avoidance mechanisms frequently are alterations in morphology that reduce evapotranspiration and conserve water. These may include deeper roots, leaf surface modifications (wax and pubescence), leaf orientation, and leaf senescence (Chaves, 1991; Hsiao, 1973; Jones and Corlett, 1992). Tolerance mechanisms enable the plant to protect the water status of critical tissues, such as the apical meristem, and mainly include osmotic adjustment (Bray, 1993). Leaves of forage grasses typically roll or fold to reduce the transpiring leaf surface exposed to the sun (Redmann, 1985). Hardy er d.(1995) examined the leaf anatomy of C, meadow and range grasses and C, range grasses and observed that the leaves of all C, grasses examined rolled or folded adaxially such that the adaxial leaf surface was completely enclosed during extreme water stress. The C, grasses showed more variability in leaf modifications with folding, rolling, or twisting of leaves most common; however, some C, grasses did not modify their leaf display in response to stress. Water stress reduces dry matter yield of forages primarily by limiting leaf area development (Ludlow and Ng, 1977; Ludlow er al., 1980; Slatyer, 1974). Van Loo (1992) partitioned leaf area expansion in perennial ryegrass (Loliurn perenne L.) into leaf elongation rate, leaf appearance rate, specific leaf area, and tillering components, and observed that tillering rate was limited by water stress principally by a reduction in leaf appearance rate. The reduction in leaf elongation rate was speculated to be a result of loss of turgor (because of only partial osmotic adjustment) andlor alteration of cell wall extensibility in response to a hormonal signal from the roots as suggested by Davies and Zhang (1991). Neumann (1995) questioned the assumption that the loss of cellular turgor pressure in response to water stress is the principal cause of growth inhibition under water stress. Neumann’s alternative hypothesis is that cell wall adjustment (hardening or softening of the cell wall), which results in smaller mature cells, is primarily responsible.
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Onillon et al. (1995) demonstratedthat yield reduction in water-stressed tall fescue (Festuca arundinacea Schreb.) resulted principally from reduced leaf area expansion and consequent loss of light harvesting surface. Fescue did not develop deeper roots in response to water deficit, and C partitioning between root and shoot was unchanged probably because root and shoot growth were reduced similarly. Spollen and Nelson (1994) induced water stress in tall fescue in the growth chamber by withholding water for 5 days, This changed the spatial distribution of growth in the basal meristem of the elongating leaf blade by shortening the growth zone from 25 to 15 mm and stopping leaf elongation by 4 days. Water stress increased the dry matter and hexose content in the leaf growth zone but decreased the fructan content. Sucrose and hexoses contributed more as osmolytes to osmotic adjustment than did fructan. They suggested that altered C metabolism as well as a limited supply of water to expanding cells reduced leaf growth. In contrast, Parrish and Wolf (1983) reported that leaf elongation rate in tall fescue was responsive solely to water uptake and flux into the elongating leaf blade. Osmotic adjustment, the ability of the plant to accumulate solute molecules that reduce osmotic potential of the cell sap, is an important physiological mechanism for dealing with water stress (Bray, 1993). In the greenhouse, Barker et al. (1993) observed that the C, grasses indiangrass [Sorghastrum nutans (L.) Nash], big bluestem (Andropogon gerardii Vitman), and switchgrass (Panicum virgatum L.) adjusted osmotically to water deficit by 0.13421 MPa, whereas the C, grasses smooth bromegrass (Bromus inemis Leyss.) and reed canarygrass (Phaluris arundinacea L.) did not adjust. In the field, both C , and C, grasses adjusted osmotically during 24 days of drought, but the extent of adjustment was greater in the C, grasses (average of 1.05 MPa) than in the C, grasses (average of 0.58 MPa). The osmotic adjustment in the C, grasses occurred within the first 14 days, whereas the C , grasses adjusted osmotically throughout the experiment. The C, grasses were able to maintain some turgor despite less osmotic adjustment by maintaining more flexible cell walls as evidenced by a lower bulk modulus of cell wall elasticity. Drought induces retranslocation of N from shoots to roots and rhizomes of perennial C, grasses (Heckathorn and DeLucia, 1994).The resulting reduced concentration of N in leaves limits photosynthesis during recovery growth. Heckathorn and DeLucia (1994) hypothesized that the N retranslocation mechanism served to limit the loss of N in aboveground organs when soil N was less available and photosynthesis inhibited.
B. EFFECTSON PHOTOSYNTHESIS Moderate water-deficit stress reduces photosynthesis primarily by inducing stornatal closure (stomatal limitation; Chaves, 1991). Nonstomatal factors have
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been implicated in causing reduced photosynthesis under severe water deficits. Antolin and Sanchez-Diaz (1993) measured the photosynthetic response and in vifro ribulose bisphosphate carboxylase (RUBISCO) activity in alfalfa (Medicago sativa L.) exposed to increasing water deficit and found evidence for stomata1and nonstomatal effects.The intercellularCO, concentration (Ci)of alfalfa leaves, calculated from gas-exchange measurements, remained constant in control and water-stressed plants. Further evidence for nonstomatal limitations were (a) reduced C,-saturated photosynthesis during water stress, (b) only partial recovery of photosynthesis and light response after rehydration, (c) an increased CO, compensation point with decreased leaf water potential, and (d) stressed plants had a reduced quantum yield (energy required to fix a unit of CO,) as measured by in v i m RUBISCO activity (in the absence of epidermis and stomata). Activated oxygen compounds, such as H,O,, 0,-, and OH, may accumulate during water-deficit stress and damage the photosynthetic apparatus. Superoxide dismutase (SOD) and ascorbate peroxidase along with the antioxidants ascorbic acid and glutathione act to prevent oxidative damage in plants (Allen, 1995). Irigoyen et al. ( 1992) measured SOD, catalase, and peroxidase levels (enzymes associated with 0 metabolism and which may alleviate damage) and levels of ethylene and malondialdehyde (indicating lipid peroxidation) in leaves of alfalfa plants stressed at several water-deficit levels. Water deficit reduced both photosynthesis and transpiration; however, transpiration was reduced more relative to photosynthesis.At moderate stress levels (- 1.6 MPa leaf water potential), levels of H,O,, ethylene, and malondialdehyde increased but were less at lower leaf water potentials. Activity of SOD was maintained during water deficit, whereas catalase activity varied inconsistently as water deficit increased. Peroxidase activity decreased curvilinearly with increasing stress. These results indicated that oxygen free radicals had little direct effect on the photosynthetic apparatus of severely stressed alfalfa leaves. In contrast, Price and Hendry (1991) found that oxidative molecular damage in several grasses was initiated in the chloroplasts and caused a cascade of damaging effects including chlorophyll destruction, lipid peroxidation, and protein loss.
C. TALL FESCUEAND ENDOPHYTE Mutualistic relationships between plants and microbes modify the physiology of stress resistance in forages. Infection of tall fescue with an endophytic fungus (Acremonium coenophialum Morgan Jones and Gams) reduces the weight gain and disrupts the metabolism of livestock feeding on the infected forage (Joost, 1995). Ironically, infection with the fungus also confers a resistance to several biotic and abiotic stresses to the plant and enables fescue to persist for many years (Clay, 1990). Infection has been shown to affect the water-stress tolerance of tall fescue
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by inducing avoidance mechanisms such as leaf rolling, increased leaf thickness, leaf senescence, reduced leaf extension, and stomata1closure (Arachavaleta et al., 1989; Belesky et al., 1989; Richardson et af., 1993). White et af. (1992) did not find evidence for an endophyte-mediated drought tolerance in tall fescue. Elmi and West (1995), however, demonstrated an increase in the level of osmotic adjustment in basal leaf meristems and leaf blades of water-stressed infected plants compared with water-stressed noninfected plants, which could increase plant survival during long-term drought stress. The physiology of these responses has been reviewed by Bacon (1993) and West (1994).
D. WATER-DEFICIT EFFECTSON FORAGEQUALITY Water deficit frequently reduces forage yields; however, because the total value of a forage crop also depends on its quality, effects of water-deficit stress on forage quality are of interest. Drought has been shown to reduce fiber concentrations and increase the digestibility of legumes (Vough and Marten, 1971; Halim et al., 1989; Peterson etal., 1992; Petit etal., 1992) and grasses (Bittman etal., 1988; Clark and Lugg, 1986; Sheaffer et al., 1992). The changes in forage composition and quality were most often the result of reduced plant maturity and increased ratio of leaf mass (of higher quality) to stem mass (often of lower quality) (Buxton and Fales, 1994). Pitman et al. (1981), however, found evidence for a direct effect of water deficit on the in vitro digestibility of stems and leaf blades of kleingrass (Panicum coloraturn L.). As xylem pressure potential declined from -0.2 to - 1.1 MPa, leaf digestibility was reduced by 19% and stem digestibility by 32%. A companion study indicated that stressed leaves and stems had an increased proportion of cell walls and increased lignification (Pitman et al., 1982).
E. SEEDLING ESTABLISHMENT IN GRASSES Establishment of forage crops is the most critical phase of perennial forage crop production even though it accounts for a brief period in the total life of the stand. Most forages are small seeded and thus planted at depths less than 25 mm. Forage seedlings are particularly vulnerable to water deficit because the extremes of water availability occur at the soil surface (Osmond et ul., 1987). The seminal root system of perennial grass seedlings originates at the depth of planting and functions for a short time (Fig. 1). It supplies water to the developing seedling for a short time and is limited by the hydraulic conductivity of the subcoleoptile internode (Hyder et al., 1971; Wilson et al., 1976; Redmann and Qi, 1992). The permanent, adventitious root system forms at the coleoptilar node, which is at or above the soil surface in panicoid grasses (Tischler and Voigt, 1987). In festucoid
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Figure 1 Diagram of festucoid (A) and panicoid (B) seedlings indicating placement of the crown and origin of adventitious and seminal roots. In seedlings of panicoid grasses, the subcoleoptile internode elongates, placing the coleoptilar node and crown (from which adventitious roots develop) near the soil surface, whereas in festucoid grasses the coleoptilar node and crown remain at or near planting depth. Diagram and terminology based on Hyder (1974). Newman and Moser (1988). and Ries and Hofmann (1991).
grasses, only the coleoptile elongates and the crown node remains at the seeding depth; thus, the adventitious roots form deeper in the soil compared with panicoid grasses. In blue grama [Boufelouagrucilis (H.B.K.) Lag. ex Steud] grown in a semiarid environment, the maximum leaf area that could be supported by the seminal root system occurred 60-70 days after seedling emergence (Wilson et al., 1976).The cross-sectionalarea of the subcoleoptileinternode was about five times less than that of an adventitious root and could supply only 1 to 2 ml of water per day compared with 5-10 ml per day for an adventitious root. Without an adventitious root system, the seedlings died after 4 months. Newman and Moser (1988) determined that adventitious root development did not occur until the third leaf stage (about 15-24 days after emergence) for many species of the Andropogoneae tribe. Tischler et al. (1989) noted adventitiousroot developmentin kleingrass within 1 week under well-watered conditions in the growth chamber. The subcoleoptileinternode usually stops elongating when the coleoptile tip intercepts and transmits light to a putative phytochrome system in the crown (a photomorphological response; Tischler and Voigt, 1993). This occurs about 3-5 days
M. A. SANDERSON ET AL. Table I Subcoleoptile Internode Length in Festucoid and Panicoid Grasses Type and species
Subcoleoptile internode length (mm)
Festucoid Agropyron daysystachuma Agropyron desertorurn" Leymus angustusa Pascopyrum smirhiib Bromus inermis
0 0 0 0.8
Panicoid Andropogon scopariusa Boureloua curtipendulaU Boureloua curtipendula Panicum virgatumc Panicum colorarumC Andropogon gerardii'
12 11 24
"Data from Redmann and Qi (1992). Seeds were planted 15 mm deep and grown for 3 weeks in the greenhouse. bData from Ries and Hofmann (1991). Seeds were planted 25 mm deep and grown for 7 days in the growth chamber. 'Data from Tischler and Voigt (1993). Seeds were planted 5 m m deep and grown for 10 days at low light (1.5 pmol m-* SKI).
after germination (Tischler and Voigt, 1996). In some grasses, however, signal transduction to the subcoleoptile internode does not occur or is delayed, and the internode continues to elongate and elevates the crown node above the soil surface. This may occur during overcast days, in seedbeds with large amounts of residue, or in tall stubble. This response has been noted in sideoats grama [Bouteloua curtipendula (Michx.) Torr.], blue grama, kleingrass, switchgrass, and other species (Olmsted, 1941; Hyder et al., 1971; Tischler and Voigt, 1996; Redmann and Qi, 1992; Table I). The transition from seminal roots to adventitious roots is a crucial stage in seedling development of perennial grasses. Successful establishment of the adventitious root system requires moist soil for about 5-7 days (Wilson and Briske, 1979; Newman and Moser, 1988). If the soil surface is dry or the crown node is elevated above the soil surface by the subcoleoptile internode, the adventitious roots may fail to develop and the seedling will die. Tischler and Voigt (1993) hypothesized that reduced subcoleoptile internode elongation would enhance survival of some panicoid grasses by placing the crown node deeper in the soil, thus enabling adventitious root development in moist soil. Qi and Redmann (1 993) compared seedling survival of several C, and C, perennial grasses at different water stress levels (-0.5, -1.0, and - 1.5 MPa). The C, grasses had better seedling survival (average of 14% seedling mortality at
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- 1.5 MPa) than C, grasses (average of 63% mortality at - 1.5 MPa). They associated greater seedling survival in the C, grasses with a larger seed size and more vigorous seedlings and poor survival in the C, grasses with seedling morphology and reliance on seminal roots for water uptake. A shorter subcoleoptile internode may also improve water transport from seminal roots to shoots. Redmann and Qi (1992) measured the diameter of xylem vessels in seedlings of warm-season grasses that emerged from different planting depths. Vessel diameter was relatively constant, whereas the subcoleoptile internode was longer at deeper planting depths increasing the path length for water transport from the root to the shoot and reducing hydraulic conductivity. Hence, reducing subcoleoptile internode elongation would reduce path length and enhance water transport from root to shoot. Tischler and Voigt (1993) described a system for screening seedlings for crown node elevation and have selected kleingrass and switchgrass germ plasm for reduced and enhanced subcoleoptile internode elongation. Populations with greater subcoleoptile internode elongation and with little or no elongation have been developed through three cycles of divergent selection (Tischler and Voigt, 1995; Tischler et al., 1996).The value of this selection remains to be determined in the field. The literature on water stress and plant water relations is vast. Losch (1995) stated that more than 2400 reports on plant water relations appeared in the literature during 1992and 1993.The physiological mechanisms and photomorphologicalresponses involved in establishmentof warm-season perennial grasses, however, require a greater understanding. Given that establishment is the most critical phase of perennial forage crop production, research is needed to enable the development of robust, rapidly establishing cultivars and to provide insights into seedbed ecology to improve management.
III. DEFOLIATION STRESS Perennial forages are harvested (defoliated) once or more during the growing season. Removing the aboveground phytomass places a large stress on the stubble, roots, and rhizomes by depriving them partially or totally of C, whereas respiration continues. Under severe defoliation, the plant may enter into a negative C balance. Forage plants recover from this C loss through immediate (e.g., reduction in N, fixation and root growth) and long-term (e.g., rebuilding leaf area) responses.
A. WHOLE-PLANT RESPONSES The defoliation stress encountered by a forage plant depends on (a) intensity of defoliation; (b) the type of tissue removed, whether meristematic and physiologic
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age; (c) frequency of defoliation, whether in discrete well-spaced events or continuous removal; (d) timing of defoliation; and (e) whether stresses or competition have occurred before, during, or after the defoliation (Richards, 1993). Removal of young, photosynthetically active leaves affects the plant more, in terms of photosynthesis, than loss of older, shaded leaves of a lower photosynthetic capacity (Gold and Caldwell, 1989a,b, 1990). Root growth stops quickly after defoliation and fine roots may die (Luo et al., 1995; Jarvis and MacDuff, 1989). Respiration and nutrient uptake also decline quickly after defoliation. These responses, however, may be tempered by the age of the plant and availability of resources. Root growth is also affected by the frequency and severity of defoliation. Alcordo et al. (1991) clipped stargrass (Cynodon nlemfuensis Vanderyst var. nlemfuensis) at several plant and stubble height combinations and observed that root mass accumulation was reduced by up to 97% with severe defoliation compared with an unclipped control. Nitrogen fixation in legumes is greatly reduced or ceases quickly after defoliation (Hartwig et al., 1987; Denison et al., 1992; Kang and Brink, 1995). Hartwig and Nosberger (1994) hypothesized that continuation of N, fixation causes N compounds to accumulate in the nodules after defoliation and reduces the aboveground demand for N compounds (reduced N sink strength). These N compounds trigger an increase in resistance to 0, diffusion into the nodules resulting in reduced nodule respiration and reduced nitrogenase activity. What triggers the increase in 0, diffusion resistance is unknown and confirmation of the hypothesis awaits further evidence. These immediateresponses to defoliation are followed by the long-term process of recovering a positive C balance, metabolic adjustment of the remaining organs, and rebuilding the photosynthetic area (Richards, 1993).Metabolic adjustment includes an increase in photosynthetic rate of the remaining leaves. Compensatory photosynthesis (defined as increased photosynthesis in leaves of defoliated plants relative to leaves of a similar age on undefoliated plants) has been observed in the remaining leaves of defoliated plants (Nowak and Caldwell, 1984;Baysdorfer and Basham, 1985) and may be related to a delay or halt in the normal ontogenetic decline of photosynthesis in leaves (Nowak and Caldwell, 1984; Wardlaw, 1990) or exposure of shaded leaves to greater levels of light or other resources (N and water) becoming more available to the remaining leaves (Pearcy et al., 1987). Compensatory photosynthesis may be induced by increases in leaf N, carboxylase activity and amount, electron transport, or stomata1 conductance (Briske and Richards, 1995). Rebuilding the photosynthetic area depends on the plant growth form, location and activity of remaining meristems, and morphological plasticity of the plant (Chapman and Lemaire. 1993). In grasses, these characteristics include bunchgrass versus sod-forming (stoloniferous or rhizomatous species) plants, timing of tiller emergence and apical elevation (synchronous or asynchronous tillering), and
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proportion of reproductive versus vegetative culms produced. Growth form variations among species, including meristem location and activity, are important considerations for management of forages to minimize plant stress after defoliation (Briske and Richards, 1995). For example, stoloniferous and rhizomatous grasses such as bermudagrass (Cynodon dactylon L. Pers.) that maintain many active meristems at axillary buds on stem bases and at nodes of stolons orrhizomes (Dong and de Kroon, 1994) may be more tolerant of close, frequent defoliation than a bunchgrass such as switchgrass. Switchgrass elevates the growing point well above ground during early vegetative growth (Sanderson and Wolf, 1995), has a high proportion of reproductive to vegetative tillers, and is sensitive to defoliation (George and Oberman, 1989) because new growth must occur from crown buds or aerial axillary meristems (Brejda et al., 1994; Hafercamp and Copeland, 1984). If all tillers on a bunchgrass plant form and develop at once (synchronous tillering), then defoliation may remove all active meristems (Culvenor, 1993, 1994). Similarly, in legumes, there is a range of architectures that enable plants to avoid defoliation stress. Upright, crown formers such as alfalfa may be vulnerable to intensive or poorly timed defoliation, whereas stolon formers such as white clover (Trifoliurnrepens L.) avoid stress resulting from frequent and intensive defoliation because of the location and abundance of current and potentially active meristems (Forde et al., 1989). In tropical legumes, there may be even greater diversity in morphologies (Kretschmer, 1989). Defoliation management of legume seedlings may also help minimize stress and improve seedling establishment. Kang el al. (1995) recommended that white clover seedlings not be defoliated until at least four trifoliolate leaves have formed and that subsequent clipping be delayed to improve seedling survival.
B. REMOBILIZATION OF CARBON AND NITROGEN New C and N compounds are preferentially allocated to active meristems in the shoot after defoliation (Wardlaw, 1990; Baysdorfer and Basham, 1985). The active aboveground meristems are stronger sinks than the roots and enable the plant to recover quickly from defoliation stress. This imbalance in sink strength is maintained until the amount of leaf area is large enough to meet the demands of the active sinks. Habben and Volenec (1990) demonstrated that starch stored in alfalfa taproots was used for shoot growth and root respiration for 14 days following defoliation. After 14 days, the taproot became a sink for C and by 28 days after defoliation starch again accumulated to high levels. Breakdown and synthesis of starch grains were spatially separated in the taproots of alfalfa during regrowth. Starch grains near the vascular cambium were used before those near the center of the taproot. Habben and Volenec ( 1990)concluded that during the early stage of carbohydrate
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remobilization starch was degraded first in the taproot bark and in medullary ray cells near the bark phloem, then in ray cells near the center of the taproot. Boyce et al. (1992) showed that endoamylase activity in alfalfa taproots was positively related with starch degradation during shoot regrowth. Kallenbach et al. (1995) hypothesized that day length regulated carbohydrate metabolism during summer regrowth of sainfoin (Onobrychis viciifolia Scop.), which has poor regrowth and persistence during summer. Although day length altered plant form (plants were taller and had more blooms under long days), there was no effect on plant yield, total nonstructural carbohydrate in root, enzyme activity (a-and P-amylase, aglucosidase, and starch-debranchingenzymes) or metabolic heat rates of the whole plant. In many temperate grasses, fructan is the storage carbohydrate remobilized for regrowth (Pollock and Cairns, 1991). Prud’homme er al. (1992) demonstrated that perennial ryegrass remobilized carbohydrates and soluble proteins from the stubble and roots to newly formed leaves during the first 6 days of regrowth. During Days 6-29, carbohydrates and proteins were replenished to levels present before cutting. These changes in carbohydrate levels were associated with changes in enzyme activities. During early regrowth, activities of anabolic enzymes declined, whereas activities of hydrolyzing enzymes increased. During carbohydrate replenishment, this pattern was reversed. Golovko and Tabalenkova (1994) demonstrated active remobilization of stored carbohydrate from stubble of annual ryegrass (Lolium multijlorum Lam.) and reported that in defoliated plants reserve carbohydrate was used only during regrowth and was not used at all in nondefoliated plants. The importance of reserve carbohydrates in regrowth has been questioned (Volenec and Nelson, 1994). Volenec (1985) observed that the rate and extent of leaf area development and stem extension during regrowth of alfalfa did not vary directly with root carbohydrate reserve levels. Hall et al. (1988) reported that even though drought-stressed alfalfa had higher concentrations of storage carbohydrates in the roots compared with nonstressed plants, there was no increase in regrowth yield of stressed plants versus nonstressed plants. Boyce and Volenec (1992) used high- and low-starch lines of alfalfa to show that shoot regrowth was not related to starch levels. Richards and Caldwell (1985) noted that the number and activity of meristems remaining after defoliation was more important than the level of carbohydrate reserve in the crown during regrowth of crested wheatgrass and bluebunch wheatgrass [Pseudoroegeneriaspicara (Pursh) A. Love]. Busso et al. (1990) demonstrated that regrowth of drought-stressed wheatgrasses was positively correlated with carbohydrate pools; however, regrowth was enhanced only when there were active meristems available. New evidence indicates that N reserves may be as important as C reserves during regrowth. Ta et al. (1990) found that plant respiration consumed most of the C stores in roots of defoliated 8-week-old alfalfa and that one-fourth of the N re-
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serves in the roots were exported to support shoot regrowth. Lemaire et al. (1992) reported that up to 40 kg ha- * of N was remobilized from alfalfa taproots and exported to support aerial regrowth. Hendershot and Volenec (1993b) determined that specific pools of N in the alfalfa taproot were used for regrowth after cutting. Aspartate and asparagine were the most prevalent amino acids in taproots and along with buffer-soluble proteins decreased greatly in concentration after defoliation. These N compounds were then replenished during shoot regrowth. The amino-N compounds were postulated to serve as readily available forms of N, whereas the proteins may be a long-term storage form. Ourry et al. (1994) generated alfalfa plants with similar crown and root dry weights but that had either high starch-low N concentrations or low starch-high N concentrations and found that the highest plant yields during regrowth occurred with high tissue N concentrations despite low starch levels. Nitrogen uptake and remobilization and shoot dry weight during regrowth were more closely related to the amount of N remaining in the source organs before clipping than to C reserves. Nitrogen compounds have also been shown to be very important in regrowth of grasses (Thornton et al., 1994; Jarvis and MacDuff, 1989; MacDuff et al., 1989). These recent advances in understanding the role of carbohydrates and plant morphology have shifted the emphasis of defoliation management based solely on optimizing or maximizing organic reserves to a recognition of the concept of morphological plasticity and the importance of maintaining active meristems that may capitalize on stored C and N compounds. Indeed, Kemp and Culvenor (1994) stated that defoliation tolerance is best enhanced by maintaining a high density of plants and tillers that have low growing points so that regrowth is rapid after defoliation or in unfavorable environmental conditions.
Tv. LOWLIGHT Leaf area development, growth rate, and yield of forages vary directly with the amount of sunlight intercepted by the canopy (Gifford et al., 1984; Lawlor, 1995). Light regulates plant growth and development via informational signals detected by phytochromes (Quail et al., 1995; Smith, 1995; Hock, 1995). The amount of sunlight intercepted by a forage plant may be reduced by the cropping system [e.g., woodland pastures (Mordelet, 1993), agroforestry systems (Pearson and Ison, 1987), or grass-legume mixtures (Beuselinck et al., 1994)],canopy structure [leaf erectness and tiller density (Rhodes, 1973)], litter accumulation (Tilman and Wedin, 1991),and climate. The responses to low-light stress generally include an increase in plant leaf area to maximize light interception and changes in physiological processes to enhance the efficiency of C utilization.
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A. SHADERESPONSES In forage grasses, responses to reduced light (shade) include larger leaves with fewer mesophyll cells and stomata per unit leaf area, more intercellular air space, higher leaf area ratios (LAR), and reduced specific leaf weight (SLW) (Allard et al., 1991a; Kephart et al., 1992). The increased leaf area is the result of longer leaves because of an increased duration of leaf elongation (Allard et al., 1991a). Leaves that develop in shade also have a reduced photosynthetic rate capacity (Evans, 1993a; Labhart et al., 1983). Kephart er al. (1992) observed that C, and C, grasses responded similarly to shade in terms of morphological adaptations (e.g., reduced SLW and increased LAR); however, C exchange rate (CER, per unit leaf area) and crop growth rate of C, grasses were reduced more by shade than C, grasses. Allard et al. (1991b) also showed a reduced CER (per unit leaf area) for shaded tall fescue along with a reduced stomata1 conductance and suggested that leaves adapted to shade by changes in both anatomical and physiological characteristics. They also determined the response of CER to light and CO, and found that the initial slope of the response curve did not differ between shade treatments, but maximal responses were less for shade-grown leaves than for leaves grown in full sun. This suggested that the efficiencies of photosynthetic reactions at low levels of input were not affected by light level during leaf development, but that the capacity of the processes was affected. Leaves in low-light environments undergo photosynthetic acclimation with a reduced photosynthetic capacity per unit of chlorophyll (Evans, 1993a,b). Leaves acclimated to low-light environments redistribute N to thylakoid membranes to maintain a constant ratio of photosynthetic capacity to total leaf N concentration, which facilitates maximum light absorption and optimizes photosynthesis in a range of light environments (Evans, 1989). Reduced light interception also influences the kinetics of leaf growth and assimilatepartitioning.Schnyder and Nelson (1989) noted that leaf blades of tall fescue grown in low versus high (60 vs 300 pmol m-’ s-l photosynthetic photon flux density) continuous light had 33% greater leaf elongation rate, a longer elongation zone, and a decreased rate of deposition of water-soluble carbohydrate. Fructan accounted for 64%of water-soluble carbohydrate deposition (25% of dry matter import into the elongation zone of the leaf blade) indicating that the elongation zone was a strong sink even at low irradiance. Sanderson and Nelson (1995) showed that reducing light in a stepwise manner resulted in longer leaves with a larger area and lower SLW, a greater leaf elongation rate, and reduced dry matter deposition in high yield per tiller and low yield per tiller genotypes of tall fescue (Table 11; Fig. 2). Increasing light at graded levels reversed these responses. Leaf elongation rate was severely reduced in darkness and dry matter deposition was stopped. The greater leaf elongation rate at low light was due to a longer zone of cell elongation in the leaf blade meristem. The data indicated that the longitudinal growth rates and spatial distribution of growth in leaf blades of tall fescue were
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Table I1 Morphological and Kinetic Responses of Tall Fescue Leaf Blades to Decreasing or Increasing Light in the Growth Chamber ~
Irradiance (pmol PPFD m-* Decreasing light 550 190 50 0 Increasing light 50 130 400
Leaf area s-I)
Specific leaf weight (mg m-’)
Leaf elongation rate (mm h-I)
I74 180 195 218
6. I 6.4 6.3 6.4
57.3 42.8 26.3 26.3
0.62 0.60 0.88 0.39
294 321 227
5.3 6.0 5.8
20.1 35.9 48.0
0.86 0.88 0.68
Leaf length (mm)
1089 1167 1252 I365 I573 1998 1330
“Leaf elongation rate data from Sanderson and Nelson (1995).Leaf dimension data from M. Sanderson and C. Nelson (unpublished data).
nearly quantitatively reversible with increases or decreases in light, similar to the photosynthetic and specific leaf area responses of annual ryegrass to alternate low and high light (Prioul et al., 1980a,b). Legumes growing in a mixture with grasses may respond to the plant canopy by placing leaves in a favorable light environment. Woledge et al. (1992) found that short and tall (small- and large-leaved, respectively) cultivars of white clover displayed leaves in the upper canopy of tall- and short-stature grasses to maximize light interception. Tolerance of some warm-season grass seedlings to shaded or high-light habitats may be related to the type of C, photosynthetic pathway used by the plant. Veenendaal etal. (1993) noted that seedlings of grasses with the phosphoenolpyruvate carboxykinase (PCK) pathway emerged and were more prevalent in shaded areas (i.e., under tree canopies), whereas in full sun, seedlings with the NAD-dependent malic enzyme pathway were more prevalent. They noted that PCK-type grasses occurred most often in moist habitats and speculated that the shade of the tree canopy reduced heat and drought stress resulting from direct sunlight. Forage quality of both C, and C, grasses was increased by shade treatments with a small decrease in fiber concentration,a small increase in digestibility,and a large increase in N concentration (Kephart and Buxton, 1993). The authors speculated that the reduced fiber concentration in shaded grasses resulted from dilution by accumulated N and perhaps because of a limited supply of photosynthate. Samarakoon er al. (1990) also reported small increases in digestibility of tropical grasses with increasing shade. Ellen and Van Oene (1989), however, showed a small
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186 0 7
PHOTOSYNTHETIC PHOTON FLUX DENSITY (umol Figure 2 Rates of dry matter deposition within the growth zone (basal 30 mm) of tall fescue leaf blades in response to graded levels of light. Data are average rates for the entire growth zone. HYT, high yield per tiller genotype: LYT, low yield per tiller genotype. Source: Sanderson and Nelson (1995).
increase in cell wall constituents and a large decrease in water-soluble carbohydrates of barley (Hordeum distichurn L.) in response to low light. Buxton and Fales (1994) noted contradicting reports of shade on forage quality and concluded that most effects of shade on forage quality were small.
B. LIGHTQUALITY Changes in light quality result in photomorphogenic changes in forages. The plant canopy not only intercepts photosynthetically active radiation (PAR) (400-700 nm wavelengths) and reduces exponentially the quantity of light that
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reaches the base of the canopy (Evans, 1993a) but also selectively absorbs red wavelengths (approximately 660 nm) more than far-red wavelengths (approximately 730 nm) such that the red:far-red ratio of light decreases toward the base of the canopy (Holmes and Smith, 1977). Common responses to altered light quality include elongation of petioles, leaves, and stems and reduced tillering in grasses and branching in legumes (BallerC et al., 1995). Robin et al. (1994) isolated the apical bud on the main stolon of white clover and exposed it to light with red:farred ratios of 2.1, 1.6, or 0.25 without affecting the amount of PAR received by the apical bud or the remainder of the plant. Light with a reduced red:far-red ratio delayed branch appearance by 0.5 phyllochrons, which subsequently resulted in a reduced number of primary branches on the plant. Frank and Hofmann (1994) demonstrated that defoliation management of perennial cool-season grasses affects the red:far-red light ratio in the canopy and stem density. Management that resulted in increased standing forage (e.g., exclusion of grazing) reduced the red:far-red ratio at the base of the plant canopy and reduced the number of stems per unit area. Removal of forage by grazing or haying increased the red:far-red ratio at the canopy base and increased stem density in the stand. As discussed under Section II,E (Seedling Establishment in Grasses), light quantity and quality affect the physiology and morphology of grass seedlings during establishment. Seedlings of kleingrass and switchgrass selected for increased subcoleoptile internode elongation required a much greater light level to inhibit crown node elevation than did seedlings selected for reduced subcoleoptile internode elongation or parent germ plasm (Elbersen et al., 1995). Preliminary data suggest that selection for reduced subcoleoptile internode elongation has increased the sensitivity of the phytochrome A/B system in the seedlings.
V NUTRIENTSTRESS Nutrient stresses in forage plants can result from either deficiency or excess. A common response to nutrient deficiency stress is a decline in growth rate and a decline in the rate at which all resources are acquired (Chapin, 1991). Other responses may include an increased ability to absorb nutrients, perhaps through altered activity or number of ion-specific carriers in root cells, increased partitioning of dry matter to roots relative to shoots, remobilization of tissue nutrients, reduced photosynthesis, and hormonal responses (Chapin, 1991).
A. NITROGEN Nitrogen is the largest fertilizer input in forage systems (Muchovej and Rechcigl, 1994). Nitrogen deficiency results in reduced photosynthesis (Woledge and
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Pearse, 1985; Bowman, 1991), reduced plant growth, and chlorosis (Fernandes and Rossiello, 1995). Nitrogen deficiency reduces cell size, volume, and protein content and reduces the number and size of chloroplasts (Nhtr, 1992). In perennial grasses, reduced leaf elongation associated with N limitation (Pilbeam, 1992; Volenec and Nelson, 1983)results from reduced cell division and not from reduced cell elongation (MacAdam et al., 1989). Gastal and Nelson (1994) examined N deposition in the growth zone of elongating tall fescue leaves and found that the rate of N deposition was greater in plants grown with high N than in those transferred to no N. Most of the N was found in the cell division zone and the least amount of N was found in the cell elongation zone. Gastal and Nelson (1994) speculated that synthesis of RUBISCO and other chloroplast proteins that occurs in the cell maturation zone uses recycled N from proteins manufactured during cell division. BClanger et al. (1992) showed that C partitioning to roots relative to shoots increased in N-limited tall fescue. Gastal and BClanger (1993) reported that N fertilization improved dry matter yield principally by speeding up leaf area development and increasing light interception rather than by increasing canopy photosynthesis. Nitrogen may be remobilized from other plant organs during periods of N stress (Engels and Marschner, 1995), and forage plants often rely on internal stores of N during regrowth (Ourry et al., 1994; Hendershot and Volenec, 1993b; Thornton et al., 1994). In response to a reduced N supply, perennial grasses increase fine roots at microsites of high N availability and increase root hair length and density (Boot and Mensink, 1990; Crick and Grime, 1987). The primary effect on forage quality is reduced crude protein resulting from N deficiency. Other effects of N on forage quality result from changes in plant morphology (leaf to stem ratio) or maturity. There are no direct effects of N on structural carbohydrate composition. Excess N in forage may accumulate as NO, and affect animal health. Also, protein quality may be affected by changes in soluble protein, nonprotein N, and protein degradability by the ruminant animal. Hanson et al. (1983) reported that N fertilization increased in v i m dry matter digestibility of smooth bromegrass due solely to an increased crude protein concentration and not to an increased digestibility of nonprotein components (i.e., cell walls). Sanderson and Wedin (1989) reported no effect of N fertilizer on in vitro dry matter digestibility or detergent fiber concentrations of smooth bromegrass. Nitrogen fertilization of grasses increased the concentration of N in cell walls (Sanderson and Wedin, 1989; Wilman and Wright, 1978). The increased crude protein in Nfertilized herbage is usually accompanied by a decrease in water-soluble carbohydrates (Wilman and Wright, 1983).
B. PHOSPHORUS Phosphorus functions centrally in plant metabolism as (a) a structural component of nucleic acids and phospholipids, (b) as an enzyme regulator through phos-
RESPONSES OF PERENNIAL. FORAGES T O STRESS
phorylation and dephosphorylation, (c) as a regulator and substrate in photosynthesis, and (d) as a modulator of gene transcription (Mimura, 1995). Several plant responses may improve uptake and utilization of P including altered root morphology (larger, finer, and more highly branched roots, and increased root hair density and length) to enable the plant to more fully explore and exploit soil P, altered transport of P across cell membranes, improved cellular metabolism and internal utilization of P, and alterations in the rhizosphere to increase P uptake (Gourley et al., 1993).In white clover, Gourley et al. (1993) found that larger root systems enabled greater shoot growth and accumulation of P, but they did not find evidence that P was absorbed or utilized more efficiently. In contrast, Dunlop and Gardiner (1993) demonstrated an enhanced rate of PO, uptake by PO,-deficient plants compared with phosphate-adequatewhite clover plants. They attributed the increased uptake to a difference in the stoichiometry of a H+/H,PO,- symport in the plants. Phosphorus is remobilized from older to younger leaves during deficiency, and the cell cytoplasm maintains a constant pool of inorganic P by using the vacuole as a reservoir of inorganic P (Mimura, 1995). Phosphorus deficiency reduces root development and alters root morphology (Christie, 1975),and roots may concentrate in the upper soil layer where P may be more available (Sanderson and Jones, 1992). Some forages, particularly C, grasses, develop symbiotic relationships with vesicular arbuscular mycorrhizae (VAM) that confer several advantages to plant survival (Hetrick et al., 1991). Mycorrhizal hyphae enhance P acquisition by increasing the volume of soil that can be exploited (Hetrick et al., 1990).Brejda et al. (1993) showed that mycorrhizal warmseason grasses had more and heavier tillers, increased root mass, greater tissue P concentrations and P recovery, and a lower root:shoot ratio than nonmycorrhizal controls. Root architecture influences the degree of colonization by VAM with greater colonization occurring on large-diameter roots than on small-diameter roots (Reinhardt and Miller, 1990; Hetrick et al., 1991). Association with VAM may enhance P uptake in calcareous soils in which P availability may be low. Azc6n and Barea (1 992) grew alfalfa in three calcareous soils and reported that VAM-inoculated alfalfa yielded as much as P-supplemented alfalfa and had similar P concentrations but lower Ca concentrations. They speculated that the P uptake mechanism of VAM suppressed excessive Ca uptake. Mycorrhizae infection may also enable nutrient acquisition strategies such as differential timing of P uptake during cool weather for cool-season grasses versus warm-season grasses (Bentivinga and Hetrick, 1992) or access to different pools of organic and inorganic P by mycorrhizal and nonmycorrhizal plants (Jayachandran et al., 1992). Mycorrhizal infection also influences competition among plants (Hetrick et al., 1994). In alfalfa, deficiencies of P result in reduced forage yield and may affect stand longevity. Walworth et al. (1986) speculated that plant death due to low P at establishment limited yield response to P applied 3 years later. Nelson et al. (1992) reported that P deficiency reduced alfalfa plant densities 3-5 years after establish-
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ment. Sanderson and Jones (1993) also demonstrated severely reduced yields of alfalfa under P stress; however, plant and shoot density was not affected after 3 years. They concluded that low yields at low P levels were a function of reduced yield per shoot. Petit et al. (1992) noted an increase in acid detergent fiber, acid detergent lignin, and stem length and a reduced leaf-to-stem ratio with increasing P fertilization of alfalfa grown in a cold (15°C day/9"C night) greenhouse environment but not in a warm (25"C/19"C) environment. Sanderson (1993) reported that alfalfa grown in a low P soil had less neutral detergent fiber and was more digestible than alfalfa grown with sufficient P. The differences in digestibility and fiber in both studies were likely due to plant maturity and/or plant morphology differences and not to direct effects on cell walls.
C. ACIDSOILSAND AL TOXICITY Large areas of the world have acid subsoils in which the low pH renders A1 more readily available (von Uexkiill and Mutert, 1994). Delhaize and Ryan (1995), Kochian (1995),and Foy (1992) have reviewed the physiology, genetics, and management of several crop species, including forages, on acid subsoils with aluminum toxicity potential. Aluminum injury in plants results in altered root growth, mainly affecting the root apex and restricts root proliferation and, hence, soil exploitation for nutrients and water. Mechanisms of A1 tolerance include Al exclusion through modification of the rhizosphere and internal detoxification of A1 via binding to proteins or complexing with other organic molecules.
D. CALCAREOUS SOILSAND FE-DEFICIENCY CHLOROSIS Calcareous soils can induce Fe-deficiency chlorosis in several forages including many forage legumes (Ocumpaugh er al., 1991;Gildersleeveand Ocumpaugh, 1988). Chlorotic plants have reduced rates of photosynthesis caused by reduced chlorophyll content in the leaves. Wei er al. (1994, 1995) described several characteristics of resistance to Fe-deficiency chlorosis in subterranean clover (Trifoliurn subterraneum L.) including a greater root:shoot ratio, lower concentrations of tissue P that may interact with tissue Fe, mechanisms for immobilizing Fe in the soil, reduced Fe requirements for plant metabolism, and a greater efficiency of Fe use. Plants respond to Fe deficiency by two mechanisms: altering the rhizosphere by releasing H+ to increase reduction of Fe3+(strategy I plants), and synthesis and release of a phytosiderophore to chelate Fe3+,along with an associated transport mechanism in the membrane (strategy I1 plants) (Loeppert et al., 1994).Nongrass plants primarily use strategy I, whereas grasses use strategy I1 to cope with Fe-de-
RESPONSES OF PERENNIAL, FORAGES TO STRESS
ficiency stress. Management of Fe-deficiency stress is limited to the use of resistant species or cultivars.
VI. LOW-TEMPERATURE STRESS Changes in temperature cause a pronounced effect on plant growth and productivity (Ong and Baker, 1985). Most temperate species are exposed to temperatures below the optimum for growth during winter months, and freezing or low temperatures limit persistence of tropical plants in northern latitudes or high altitudes. Chen (1994) classified low-temperature stress into chilling (10-15°C and below) and freezing (below O”C, when ice forms in tissues) stresses.
A. CHILLINGSTRESS Initially, low temperatures stop cell division and elongation (Pollock et al., 1984).The timing of the response to low temperaturesin extending cells compared with the cell cycle (Frances and Barlow, 1988) suggests that the extending cells themselves respond directly to temperature changes (Pollock and Eagles, 1988). Carbon fixation and translocation do not limit cell growth at chilling temperatures. Leaf growth in dallisgrass (Paspalum dilarutum Poir.) was more sensitive to low temperature than was photosynthesis or starch accumulation (Forde er al., 1975). Chilling-resistant plants did not reduce translocation of C at low temperatures (Berry and Raison, 1982). Simultaneous measurements of extending cell turgor, leaf temperature, and leaf extension rate suggested that unacclimated leaves of Lolium temulentum did not change in turgor pressure between 20 and 2”C, which would limit cell expansion (Pollock and Eagles, 1988). Pollock and Eagles (1988) interpreted these results to imply that the sites at which low-temperature signals were perceived and transduced were linked to and acted directly on the cell wall in affecting cell growth.
B. ACCLIMATION TO Low TEMPERATURE AND DEVELOPMENT OF FREEZING TOLERANCE The secondary effects of low temperature occur during extended exposure and increase tolerance to suboptimum temperatures. Plants that grow in suboptimum temperatures and acquire increased tolerance to freezing have “hardened.” The hardening process takes several weeks to reach a maximum in many perennial forages. Different levels of freeze tolerance may develop depending on the genotype
M. A. SANDERSON ET AL.
and hardening conditions. Dehardening may take less than 1 week and occurs without subsequent lowering of temperature or on reintroduction to warm temperatures. Acclimation results from active metabolic processes associated with changes in gene expression (Hughes and Pearce, 1988; Howarth, 1990). Changes in cellular lipids occur within hours after exposure to low temperatures (Dickens and Thompson, 1981; Lynch and Thompson, 1984) and may be very important to subsequent freeze tolerance (Webb et al., 1994; Uemura and Steponkus, 1994). Both qualitative and quantitative changes in protein populations (Uemura and Yoshida, 1984) and isozyrnic shifts (Roberts, 1974) occur during growth at low temperatures. Many of these changes function to adjust metabolism at low temperatures. Physiological adjustments to low temperature may alter plant growth. For example, growth traits of “fall-dormant” alfalfa cultivars conditioned by cool short days include a prostrate growth habit, shorter internodes, and slower regrowth in fall. These traits also correlate with increased winter hardiness (Barnes et al., 1979). Similarly, fall yield has been negatively correlated with winter survival; however, limited fall growth of fall-dormant alfalfa is correlated with a reduced yield in the spring and summer (Stout and Hall, 1989). Reciprocal grafts of falldormant and nondormant alfalfa cultivars grown in warm long days or cool short days indicated that expression of prostrate growth and shorter internodes was strongly conditioned by the plant shoot mass with some mediation by the root, whereas slower regrowth was conditioned by both shoots and roots (Heichel and Henjum, 1990). Changes in growth in response to low temperature similar to fall-dormant alfalfa have been reported in timothy (Phleum pratense L.) (Klebedsadel and Helm, 1986), white clover (Woledge and Suarez, 1983; Ollerenshaw and Baker, 1981), and berseem clover (Trifolium alexundrinium L.) (Barnes and Wilson, 1986). Similar changes were noted when abscisic acid (ABA) was applied to berseem clover at higher temperatures (Barnes and Wilson, 1986) and may mediate the changes in other species. Nitrogen fertilization during growth at low temperatures increased leaf, stolon, and petiole dry weight, leaf area, and photosynthetic rate of white clover. The number of leaves and stolons and the respiration rate at low temperature were unaffected by added N (Woledge and Suarez, 1983). In white clover, cold hardiness increases with time spent at temperatures less than 0°C under short days (Collins and Rhodes, 1995) and frost hardiness at 0.5 or 6°C increased with increased N supply (Sandli et al., 1993). Cool-season forages in colder parts of the upper south in the United States must develop some level of winter dormancy so that root and crown reserves are not depleted by top growth during brief warm spells followed by killing freezes (Ball et al., 1991) Although fall growth can indicate relative winter hardiness among divergent groups of alfalfa, no correlation with freezing tolerance was found among 15 alfalfa populations with similar fall growth habits (Bowley and McKersie, 1990).
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The type and placement of perennating organs of perennial forages affect lowtemperature survival. Perennating structures in forages include crowns and rhizomes in rhizomatous species. Both structures often have a similar freeze tolerance (Schwarz and Reaney, 1989). Subcrown internode length of 7-day-old perennial ryegrass seedlings was negatively correlated with winter survival of the same cultivars in the field (Wood and Cohen, 1983). Reduced internode length enhanced winter hardiness because the meristem was placed deeper in the soil. Rhizomatous grasses with subterranean perennating structures survived severe winters in Alaska better than timothy, which required an insulating snow cover for maximum winter survival because the crown was exposed (Klebesadel and Helm, 1986). Differences in rhizome depth among ecotypes also acount for differences in vegetative overwintering in Johnsongrass [Sorghum halepense (L.) Pers.] (Warwick et al., 1986). Water content of plants may change during growth at low temperature and affect the dessication aspects of freeze tolerance. Freeze-tolerant cultivars of timothy in Alaska had less moisture in the crown compared with nontolerant cultivars (Klebesadel and Helm, 1986). In switchgrass, however, crown moisture was not correlated with freeze tolerance (Hope and McElroy, 1990), and Bowley and McKersie (1990) found no correlation between crown moisture and freeze tolerance in alfalfa plants of the same chronological age. Genotypes vary in the time and temperature required for low-temperature hardening. Gay and Eagles (1991) modeled this process and calculated hardening kinetics. The model indicated that the perennial ryegrass cultivar Grasslands Ruanui was only 34% hardened at 14 days at 2"C, whereas the cultivar S23 was 78% hardened at 14 days, though both cultivars had similar maximal levels of freezing tolerance. Rates of deacclimation were also cultivar specific. Low-temperature hardening also depends on genotype and age. Limin and Fowler (1987) classified seedlings of fall-seeded forage grasses in Canada into three categories of risk for winter-kill. One group had less freeze tolerance than winter wheat (Triticum aestivum L.) and needed the insulation of deep snow cover for winter survival. A second group attained the same freeze tolerance as winter wheat and a minimal snow cover was needed for survival. The last group attained the greatest degree of freeze tolerance, similar to that of winter rye (Secafe cereafe L.). When seeded in the spring and well established by fall, all cultivars attained at least the freeze-tolerance level of winter wheat. Freeze tolerance of 1to 3-week-old alfalfa seedlings increased during 4 weeks of growth at 1°C and older seedlings attained a greater degree of tolerance (Cloutier et af., 1990). The amount of messenger RNA (mRNA) induced by stress was correlated with the age of alfalfa plants (Luo et af., 1992) and may account for the age dependence observed in other studies. Warm-season C, forage grasses increased in freeze tolerance with exposure to low nonfreezing temperatures and dehardened on return to warm temperatures
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(Anderson et al., 1988; D. W. Stair, unpublished data) similar to temperate grasses though usually not to the same extent. Some C, grasses in northern latitudes, however, increased their level of freezing tolerance equal to that of temperate grasses. Northern ecotypes of switchgrass have been shown to increase their freeze tolerance and survive temperatures of - 19 to -22"C, similar to timothy and alfalfa in the same location (Hope and McElroy, 1990). Four C, grasses [Distichlis stricta (Torr.) Rydb.; Sparfina gracilis Trin.; Schizachyrium scoparium (Michx.) Nash; and blue grama] reached the same degree of freeze tolerance (-27°C). The more northern species achieved these levels faster when grown at similar temperatures (Schwarz and Reaney, 1989). Freeze tolerance in alfalfa increased when exposed to 2°C for 2 weeks; however, after 2 weeks the freeze tolerance declined somewhat. Exposing plants to -2°C after the first 2 weeks at 2°C allowed acclimation to continue (Monroy et al., 1993a; Castonguay et al., 1993, 1995). ABA is associated with many stress responses (Luo et al., 1993).Application of ABA to alfalfa (Mohapatra et al., 1988) and berseem clover (Barnes and Wilson, 1986) plants increased freezing tolerance at nonhardening temperatures; however, greater freezing tolerance was acquired with low temperatures in alfalfa. Giberellic acid (GA) counteracts many effects of ABA and inhibited ABA-induced freezing tolerance in berseem clover (Barnes and Wilson, 1986). The changes in freezing tolerance may be regulated by cellular Ca movement and Ca-dependent phosphorylation of proteins. By blocking Ca2+ channels, calmodulin activity, and Ca2+-dependent protein kinase activity, Monroy et al. ( 1993b) inhibited cellular freezing tolerance acclimation. Freezing damage to cellular components results from a change in chemical potential of water during freezing. Supercooling suspension cultures of smooth bromegrass cells to -5°C produced a profile of electrolyte leakage similar to that of nonstressed samples and their viability was similar. Samples frozen at -5"C, however, showed acute electrolyte leakage characteristic of samples frozen at all temperatures, suggesting that temperature alone has little effect on cellular damage (Zhang and Willison, 1989). Winter-hardy cultivars of alfalfa progressively increased in LT,, (the lethal temperature for 50% of the population) as measured solutes increased in size from ions to macromolecules, suggesting progressive damage to the plasma membrane as the freezing temperature decreased. Nonhardy cultivars, however, showed an acute damage to the plasma membrane as evidenced by the concurrent leakage of solutes of all sizes at the same freezing temperature (Sulc et al., 1991). Cellular adjustments to growth at low temperature improve physiological functions at the prevailing temperature. Alfalfa roots maintain higher respiration, nodulation ability, and acetylene reduction levels, and activity levels of some enzymes increase more in winter-hardy cultivars of alfalfa during low-temperature growth than in less hardy cultivars (Duke and Doehlert, 1981). After a cold-induced period of growth cessation in alfalfa, nitrogenase relative efficiency was restored and
RESPONSES OF PERENNIAL FORAGES TO STRESS
nodules grew, and the temperature optimum for photosynthesis broadened in winter-hardy types (MacDowall et d., 1988). Zhang et al. (1992) observed that electrical resistance of the cytoplasm and the vacuole increased in birdsfoot trefoil (Lotus corniculatus L.) during cold acclimation.The increase in resistance was attributed to increased concentrations of sugars, which increased viscosity. At the same time, the capacitance of the plasmalemma and the vacuolar membrane decreased during cold acclimation. This was attributed to a decrease in cell size or relative increase in xylem. Enzyme stability at low temperature contributes to low-temperature survival. Krall and Edwards (1993) reported that phosphoenol pyruvate carboxylases from guinea grass lost up to 50% of activity after 60 min at O"C, whereas the enzyme from Panicum miliaceum L. maintained its activity at 0°C. This difference in stability at 0°C may result from different hydrophobic bonding in the active tetramer (Krall and Edwards, 1993). In the C, grass, Echinochloa crus-galli, several enzymes in the C fixation pathway of plants from warm environments were more affected by low temperature than the same enzymes in plants from cold environments. The most cold-labile enzyme was NADP+-malate dehydrogenase (Potvin et al., 1986). Zhang and Willison (1989) found that a freezehhaw cycle caused acute leakage immediately after thawing with subsequent chronic leakage. The only difference between treatments was the severity of the acute leakage after thawing. The length of time spent at a specific freezing temperature had no effect on electrolyte leakage. The authors suggested that the acute leakage is associated with membrane rupture and the chronic leakage is associated with altered permeability of the membrane after resealing. Activated oxygen radicals may cause damage in freezing stress and are alleviated by superoxide dismutase activity. Four alfalfa plants transformed with SOD attached to a constitutive promoter had increased regrowth after freezing stress (McKersie et al., 1993). Regrowth after freezing stress was faster for F, progeny from one of the plants that had a single functional SOD copy in the chloroplast. This suggests a protective role for SOD in freezing stress.
C. LOW-TEMPERATURE STRESSAND ORGANIC RESERVES The most commonly studied metabolites of perennial forages under cold stress are nonstructural carbohydrates.Storage carbohydrates may provide food reserves and C skeletons for conversion to sugars by the overwintering plant. An alfalfa genotype that accumulated large amounts of starch had better winter survival than a low-starch genotype. Starch decreased in both genotypes during the late acclimation period, and concentrationsof total sugars increased concomitantly (Boyce and Volenec, 1992).The decrease in starch in alfalfa crowns was correlated with
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an increase in freeze tolerance (Castonguay et al., 1995). A greater content of carbohydrate in stolons of white clover improves overwintering ability (Collins and Rhodes, 1995). Low nonfreezing temperatures increased the amount of fructans in leaves and increased the partitioning of fructans to roots of tall fescue (Prud’homme et al., 1993). Similar increases in fructan concentrations occurred in roots of Poa pratensis (Solhaug, 1991) and Agropyron and Agrosris alba (Chatterton et al., 1987).The most freeze-tolerant cultivars of timothy also had the highest levels of nonstructural carbohydrates as measured by etiolated growth of the overwintering tissue in warm temperatures (Klebesadel and Helm, 1986). Temperatures lower than 10°C may inhibit starch mobilization in leaves of tropical grasses (Shatters and West, 1995). The accumulation and translocation of soluble sugars changes in response to low temperature.A non-winter-hardy cultivar of alfalfa accumulated fructose, glucose, and maltose in stems and leaves during cold hardening, whereas a winterhardy cultivar had reduced concentrations of soluble sugars in stems and leaves (Green, 1983) and soluble sugars increased in the crown during cold hardening (Castonguay et al., 1995; Duke and Doehlert, 1981). A winter-hardy high-starch genotype of alfalfa also had higher levels of total sugars during the winter than low-starch genotypes (Boyce and Volenec, 1992).The accumulation of simple and complex nonstructural carbohydrates may be related more to decreased utilization than to photosynthetic production (Farrar, 1988). Specific sugars may protect against damage due to dessication at freezing temperatures. Differences in freeze tolerance among alfalfa cultivars were related to levels of raffinose and stachyose (Castonguay et al., 1995). Bruni and Leopold (1991) suggested that disaccharides protect membranes and proteins from desiccation by forming a glassy state that slows molecular motion and prevents damaging interactions. Sucrose may enable plants to survive desiccation by stabilizing membranes and proteins (Hoekstra e? al., 1989). Sucrose concentrations in alfalfa, however, were not correlated with maximum freezing tolerance, and glucose concentrations decreased during low-temperature hardening (Castonguay et al., 1995). Glucose may be negatively correlated with desiccation tolerance because it may participate in the Maillard reaction, which can deactivate protein and change DNA (Koster and Leopold, 1988). Total N, soluble amino-N, and buffer-soluble protein increased in alfalfa taproots during the autumn and subsequently decreased during spring regrowth. Non-winter-hardy types of alfalfa accumulated less soluble protein than winter-hardy types (Hendershot and Volenec, 1993a). Other changes may act directly in gene expression. In cold-hardy alfalfa cultivars, putrescine concentrations increased during cold hardening at 2°C and levels of spermidine remained constant, whereas concentrations of both compounds decreased in plants of similar age grown at 22°C. Spermine concentrations decreased in both temperature treatments. This trend was similar to that found in wheat under the same treatments (Nadeau et al., 1987).
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D. TISSUE CULTLTRE AND GENEEXPRESSION IN LOW-TEMPERATURE STRESS Tissue culture has been used in many studies to identify cellular mechanisms and molecular aspects of tolerance to low-temperature stress and to identify stresstolerant germ plasm. Freeze tolerance was induced by ABA in suspension cultures of smooth bromegrass (Reaney and Gusta, 1987), birdsfoot trefoil callus (Keith and McKersie, 1986), and alfalfa cell cultures (Orr et al., 1985), though kinetin had to be excluded for birds-foot trefoil and alfalfa. The degree of tolerance induced in smooth bromegrass cultures was similar to that attained by whole plants at low temperature and decreased the time needed for hardening to 7 days (Tanino et al.. 1990). Reaney et al. (1989) found that the application of GA 4, GA 7, and GA 9 blocked the ABA-induced freeze tolerance, whereas GA 3 was not effective, and that kinetin hastened the dehardening process in smooth bromegrass cell cultures. Alfalfa cell cultures treated with ABA also had slower dehardening rates than untreated cell cultures (Reaney and Gusta, 1987). Both cell expansion and division decrease at low temperatures; however, metabolic activity continues. Robertson et al. (1987) found that protein synthesis continued in bromegrass suspension cultures despite the cessation of net growth in cells at 3°C. Most major proteins synthesized in cells grown at 23°C were also detected in cells grown at 3°C. New proteins were also synthesized in cells grown at 3°C or treated with ABA, whereas synthesis of other proteins was inhibited (Robertson et al., 1988). The pattern of protein synthesis in alfalfa cell cultures was also altered by both low temperature and ABA in smooth bromegrass cell suspensions (Robertson er al., 1988) and alfalfa cell cultures (Mohapatra et al., 1988). Some overlap in the change in protein expression occurred between ABAand low temperatures, whereas some changes were specific to either treatment. One protein was regulated by ABA or low temperature and showed greater response in alfalfa cultivars that are more freezing tolerant (Mohapatra et al., 1988). In smooth bromegrass, one of the low-temperature-induced proteins was located in a membrane, whereas another was soluble in water (Robertson et al., 1988). The changes in protein and RNA populations due to low temperature were dependent on alfalfa cultivar. More rapid rates of 35Sincorporation and increased levels of protein and RNA were found in Saranac than in Anik as well as faster acclimation and deacclimation; however, Anik was more freezing tolerant at maximum acclimation (Mohapatra et al., 1987). Gatschet et al. (1994) and Anderson and Taliaferro (1995) have demonstrated freezing tolerance acclimation in warm-season C, grasses. The LT,, of bermudagrass decreased 5°C in response to low temperature and was associated with the induction of cold-regulated (COT) proteins, which are correlated with freezing tol-
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erance. These studies suggest the importance of protein expression; however, the significance of many proteins is unknown. Lee et al. (199 1) compared mRNA populations induced by ABA or low temperature in cell suspension cultures of smooth bromegrass and found that ABA induced more changes in the mRNA and protein population than did low temperature. Three of the changes were common to ABA and low temperature. Abscisic acid-induced mRNA were still present after 7 days without ABA and the LT,, of these cell cultures remained lower. The second stage of hardening in alfalfa also has associated changes in the pool of mRNA (Castonguay et al., 1993). Many of these were found to be glycine-rich proteins on translation. Some were specific to the freeze-tolerantcultivar, whereas others were altered in response to water stress and ABA. Dehardening reduced the expression of many cor mRNA. Analysis of isolated cDNAs associated with freezing tolerance induced by ABA in smooth bromegrass suspension culture showed new expression, upregulated expression, and transient expression (Lee and Chen, 1993). Genes cloned from each of these groups had homology with genes associated with sugar metabolism, osmotic stress, and protease activity. Monroy et al. (l993a) found that the increase in freeze tolerance at -2°C in alfalfa was paralleled by an increase in the expression of casl5, a cold-induced gene. Expression of this nuclear-targeted product occurred even when protein synthesis was inhibited, suggesting that expression depended on preexisting gene products. The structure of this gene was different between freezing-tolerant and freezingsensitive cultivars. Three cold acclimation specific (cas) genes have been identified in alfalfa by Mohapatra et al. (1989) and are coordinately regulated at the level of transcription. Expression of these genes was positively correlated with the degree of freeze tolerance. Other genes induced by ABA in alfalfa were induced by many environmental stresses including low temperature (Luo et al., 1992). The cDNA sequence of a cas gene from alfalfa indicated that the product was a small hydrophilic glycine-rich protein with homology to proteins associated with dessication tolerance. These transcripts accumulated slowly during cold acclimation, whereas they disappeared rapidly during cold hardening. Transcription experiments indicated that the stability of the transcript was greatly increased and may have accounted for the increased expression (Wolfraim et al., 1993). Dessication tolerance is important in freezing stress due to the loss of cellular water across the plasmalemma to the frozen extracellular matrix. Expression of a dehydrin gene was increased in smooth bromegrass acclimated in the field during the fall, smooth bromegrass cell suspension cultures treated with ABA, and bromegrass grown hydroponically at a 2°C day temperature and a -2°C night temperature for 28 days (Robertson et al., 1994). Dehardening of the plant material decreased the levels of dehydrin expression. Increases in dehydrin expression were accompanied by an increase in freeze tolerance and treatments that did not result
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in the increased expression of dehydrin genes did not show an increase in freeze tolerance.
VII. SALTSTRESS Scarcity of fresh water, competition for fresh water, and soil salinization have resulted in a need for grasses and legumes with increased salt tolerance, especially in irrigated regions of the world. Salts lower the solute potential of the external solution, presenting the plant with increased water potential gradients to overcome. Salinity reduces seed germination, stand establishment, and yield in forage grasses and legumes, but susceptibility of plants to salinity stress at different developmental stages varies among species. Plants cope with salinity stress by accumulating (primarily in tissues removed from meristems), extruding, or diluting ions, and by selective ion absorption (e.g., absorbing K in the presence of high Na).
A. SALTACCUMULATION Most plants accumulate ions in saline environments.Ando et al. (1985) reported intraspecific variation in the degree of salt accumulation, ranging from 0.32% for colored guineagrass (Panicurn coloraturn var. coloraturn) to 2.33% for Kabulabula grass [ P . colorarum var. rnakarikariense (Gossens) Van Rensb.]. Interspecific variation in accumulation exists as well. The pattern of Na and C1 uptake with an increasing level of salt differed between tropical legumes but was not related to the degree of tolerance observed (Keating et al., 1986). Shoot and root concentrations of Na and C1 were elevated under high salt treatments for St. Augustine grass (Stenoraphrurn secondturn Walt.), a moderately salt-tolerant grass, and seashore paspalum (Paspalurn vaginaturn Swartz.), a very salt-tolerant grass. Seashore paspalum also maintained higher K concentrations under salt stress. In contrast, bermudagrass maintained lower concentrations of Na and C1 under high salinity, but was less tolerant to salt (Marcum and Murdoch, 1990). High productivity under salinity stress in subterranean clover was positively correlated with restricted Na uptake in the shoot and the maintenance of high K/Na ratios (Shannon and Noble, 1995). In white clover, an extremely salt-sensitive species, salt-tolerant cultivars had lower concentrationsof Na and C1 in shoots than nontolerant cultivars, suggesting that tolerant cultivars had an improved ability to regulate uptake of both ions (Rogerset al., 1994).Plant tissues also vary in salt content. Tall wheatgrass [Thinopyrurn ponticurn (Popd.) Barkworth and Dewey] and salt-tolerant lines of crested wheatgrass had greater concentrations of K, lower Na, and lower NdK ratios in leaves than salt-intolerant lines of crested wheatgrass. Tall wheat-
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grass, however, had greater concentrationsof K, Na, C1, and lower NdK ratios in roots than crested wheatgrass (Johnson, 1991).
B. SEEDLING AND ADULTPLANTRESPONSES Increasing levels of salinity generally reduce seed germination and seedling emergence. Significant differences in germination over several saline solutions have been reported in perennial ryegrass (Horst and Dunning, 1989), subterranean clover (Shannon and Noble, 1995),and alfalfa (Al-Niemi et al., 1992).Simple correlations between alfalfa cultivars and germination in various salts indicated that germination was most affected by Na concentration; however, when all independent variables were considered,concentrationsof C1 and Mg were most important. Thus, selection in NaCl solutions should account for most of the potential gain in germination salt tolerance (Rumbaugh et af., 1993). A selected alfalfa line maintained constant germination to twice the salt osmotic pressure that the parents did. The selected line also germinated faster (Robinson et al., 1986). The level of germination of alfalfa in saline conditions was not related to postgermination performances (Al-Niemi ef al., 1992). Rhodesgrass (Chloris guyana Kunth.) populations from five cycles of selection under high salt conditions showed a significant improvement in survival and regrowth compared with unselected material. The selected material also had increased germination at moderate salt levels and decreased water use per unit leaf area. There were no apparent differences between the selected and unselected material in growth, ion content, or protein synthesis of the plants (Malkin and Waisel, 1986). An alfalfa population selected for high NaCl tolerance during germination had greater improvement in germination in NaCl than in other salt solutions and a higher germination percentage in high osmotic solutions of mannitol. The selected and parent populations did not differ in accumulation of Na or C1 during 48 h or in absorption of tritiated water during 12 h. The major physiological difference determined was that the population tolerant to low salt had a higher seed respiration rate than the population tolerant to high salt. Tolerance appeared to be due to specific ion inhibitions and osmotic tolerance and not to differences in ion uptake or imbibition rates (Allen et al., 1986). Salt tolerance at germination for seed lots of different ages from the same germ plasm source differed significantly as a percentage of nonsaline germination. Solute leakage during imbibition increased as seed age increased and was correlated with declines in germination of aged seed but not with fresh seed (Smith and Dobrenz, 1987). Degradation of plasmalemma function may allow the entry of more ions than the germinating embryo can tolerate. Examination of parental and selected alfalfa populations for different saccharides revealed no differences. Although raffinose and sucrose were higher in the seeds from the selected line, Dobrenz et af. (1993) suggested that the increase was not enough to account for the increased salt tolerance.
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Irrigation with saline water results in salt accumulating in the surface soil. Imgation with water with a conductivity of greater than 4.3 dS m-' strongly inhibited final emergence of alfalfa seedlings because of salt accumulation even though the seed had the potential to germinate in 28 dS m- water (Assadian and Miyamoto, 1987). The authors suggested that reduced emergence at seeding depths less than 5 mm resulted from reduced seed germination, whereas reduced emergence from greater depths resulted from damage to the hypocotyl caused by accumlated salts in the surface soil. Grasses and legumes vary within and among species for growth responses to salinity, often showing increased dry weight with moderate increases in Na levels (Ando et al., 1985; Ashraf et al., 1986a; Shannon and Noble, 1995). Panicum coloratum var. Bambatsi was more tolerant to salt than several tropical legumes; it sustained a 50% yield reduction at 16.4 dS m- I , whereas the best tropical legume had a 50% yield reduction at 10.6 dS m-I (Keating et al., 1986). Rhodesgrass, a halophytic forage grass, shows stimulated growth of single roots under NaCl concentrations that inhibit growth of the whole plant (Waisel, 1985). A salt-tolerant St. Augustine grass cultivar (Seville) had a 50% reduction in top growth at 28 dS m-', whereas the less tolerant cultivars exhibited a 50% reduction at 22 dS m-I. The differences between cultivars were largest at moderate salinity levels (Dudeck et al., 1993). Seville reacted to salt stress by increasing the root length, whereas a less salt-tolerant cultivar had stunted root growth under saline treatments (Meyer et al., 1989). Kallargrass [Leptochloafusca (L.) Kunth.] tolerates high salinity by secreting ions (Jeschke etal., 1995). Salt glands on its leaf blades resemble two-celled structures described in some halophytic genera of Poaceae. Secreted Na and C1 crystallizes in larger amounts than K when plants are grown under high-salt conditions (Wieneke et al., 1987). Vesicles (swollen epidermal hairs resembling the salt glands of Bouteloua sp.) occur on the leaf and sheath surfaces of kallargrass and are more common on the upper surface of leaves, whereas on the sheath they occur mainly on the lower (outer) surface. These structures may help regulate plant salt content in kallargrass (Bhatti et al., 1992a). The greatest variation in salt tolerance and Na, C1, and K uptake in kallargrass accessions came from different collection sites (Warwick and Halloran, 1991). For an accession, the internal concentrations of K, Ca, Na, and C1 in the shoots and roots were constant over a wide range of external NdCa ratios. In contrast, the shoot K and Ca and the root Ca concentrations in Panicum turgidurn remained unchanged at all external NdCa ratios, whereas the root K concentration decreased significantly at high external NdCa ratios (Ashraf and Naqvi, 1991). The Na and CI concentrations of kallargrass were higher in leaf sheath than blades and increased greatly with leaf age, whereas K concentrations were highest in young leaves and decreased with age. The authors state that K retranslocation occurs and suggested that K recycling and the use of Na to maintain turgor in old leaves is important in t? turgidum. Both Mg and Ca concentrations increased with leaf age
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(Bhatti et al., 1992b).Time-dependent secretion from leaves of kallargrass showed a large linear secretion of Na and C1 during 24 h, whereas secretion of K and Ca could not be detected before 2 and 6 h, respectively. Light stimulated the secretion of Na and C1 from the leaves and chilling the roots decreased the secretion of Na and C1 (Bhatti and Sarwar, 1993). The influence of NaCl treatment on the activity of three photorespiratory enzymes from three legumes [Cicer arietinum L. (salt sensitive), Arachis hypogaea L. (intermediate salt sensitivity), and Sesbania aculeata Poir. (salt tolerant)] was investigated. Moderate levels of NaCl inhibited the activity of 3-phosphoglycolate phosphatase from C. arietinum and A. hypogaea, whereas enzyme activity was not inhibited by NaCl in S. aculeata. Inhibition of this activity can elevate the concentration of phosphoglycolate, which is a strong inhibitor of part of the Calvin cycle. The activity of glycolate oxidase was increased slightly in C. arietinum and strongly in A. hypogaea. Salt treatment also increased the catalase activity in S. aculeata, whereas catalase activity was reduced in the other two species (Murumkar et al., 1985). The reduction of catalase activity can lead to elevated H 2 0 2 levels in conjunction with the increase in oxidase activity leading to oxidative damage in the cell. Compatible osmolytes are often used by plants to counter the chemical potential of sequestered ions in the cell. Total organic acid concentration in alfalfa nodules and roots was depressed by more than 40% in a moderately saline environment; however, lactate and amino acid concentrations increased. Proline increased the most in roots and nodules, indicating an osmoregulatory use in nodules as well as roots. Asparagine increased as well and was a major osmoregulatory component in bacteriods. The salt treatment had very little effect on other amino acids. The carbohydrate pool was increased also with pinitol increasing significantly in the cytosol and bacteroids, whereas trehalose concentration remained low (Fougere et al., 1991). Histone acetylation of alfalfa was similar in salt-tolerant and salt-sensitivecells under normal growth conditions. Exposure to short-term salt stress in salt-sensitive cells or continued growth at 1% NaCl for salt-tolerant cells resulted in large increases in multiacetylated forms of histone H4 and two forms of H3. Waterborg et al. (1989) state that the increase is an in vivo reporter suggesting an altered intranuclear ionic environment and may be an adaptive response to allow chromatin function in a more saline environment.
C. TISSUE CULTURE AND GENEEXPRESSION INSALTSTRESS Tissue culture has also been used to identify tolerance mechanisms and germ plasm tolerant to salinity. Cellular mechanisms for dealing with salinity stress may be different from
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whole-plant mechanisms and selection in vitro may not result in plants with increased salt tolerance. Young inflorescence explants of napiergrass (Penniseturn purpureurn Schumach.) exhibited delayed initiation of callus and a gradual decrease in growth with increasing salt level. Cell lines that tolerated up to 2% salt in the media were isolated and complete plants were regenerated from cell cultures tolerant to 0.5% salt (Bajaj and Gupta, 1986).Afalfa cell lines tolerant to NaCl retained their tolerance after 16 weeks of subculturingon media with no NaCI. Plants regenerated from these salt-tolerant lines exhibited somaclonal variation for salt tolerance compared with plants regenerated from control cell lines. All plants were morphologically abnormal with some extreme dwarfs. Regenerated plants from this study had unbalanced chromosome sets with one variant having 106 chromosomes. Isozyme phenotypes were also altered compared with control lines. Although the in v i m salt tolerance was maintained after regeneration for 9 of 12 regenerates, whole-plant tolerance was found only in two plants, one of which was sterile and the other did not flower (McCoy, 1987). Saline environments can alter gene regulation.A salt-inducedcDNA specific to a salt-tolerantalfalfa line had a charateristic zinc finger motif common to proteins that are nuclear transcription factors (Winicov, 1993). This will alter subsequent patterns of expression and stability. Eleven cDNA clones are induced within 2 h after exposure to high levels of salt. The decline in expression divided these genes into two groups, one returned to basal levels by 24 h and the other group between 3 and 7 days. The authors termed the rapid coordinate expression of this large number of genes an “early salt stress response” (Gulick and Dvorak, 1992). A salt-inducible cDNA from alfalfa encoded a 40-kDa cell wall protein containing a repetitive proline-rich sequence and a cysteine-rich carboxyl-terminalsequence with homology to nonspecific lipid transferases. The accumulation of this transcript in salt-tolerant alfalfa cells due to the presence of salt is primarily due to increased stability of the mRNA (Deutch and Winicov, 1995).
VIII. PLANT BREEDING FOR ABIOTIC STRESS TOLERANCE Cultivars of perennial forages have been developed with tolerance to the abiotic stresses discussed in this chapter; however, improved tolerance to abiotic stress has generally been an indirect response of selecting for superior agronomic traits such as establishment and yield among others. Recent reviews of breeding for stress tolerance (Ashraf, 1994; Gay, 1994; Hall, 1992; Noble and Rogers, 1993; Thomas, 1994) have been published in which physiological processes are discussed in detail relative to whole-plant stress tolerance. The most successful approaches to breeding perennial forages have historically used field-based evalua-
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tions to identify tolerant species, followed by selection within that species for genotypes that combine performance in stressful environments with forage quality traits (Barker and Kalton, 1989; Meyer and Funk, 1989; Busey, 1989; Beuselinck er al., 1994). Studies of plant response to abiotic stress indicate that genetic variation is available for traits such as water-use efficiency (Asay and Johnson, 1990; Barker et al., 1989; Frank, 1994; Johnson et al., 1990; Johnson and Rumbaugh, 1995; Read et al., 1993), growth under nutrient stress (Baligar et al., 1989; Edmeades er al., 1991; Lafever, 1981; Mackay et al., 1991; Wheeler et al., 1993ab), salinity tolerance (Al-Niemi et al., 1992; Ashraf er al., 1986b, 1987; Asins et al., 1993; Gregorio and Senadhira, 1993; Rumbaugh and Pendery, 1990; Smith er al., 1994), and defoliation tolerance (Brummer and Bouton, 1992;Jaindl et al., 1994; Jones et al., 1991; Smith and Bouton, 1993). Unfortunately, plant breeders have not incorporated this variation into cultivars that exhibit whole-plant stress tolerance. There are several reasons for this: (a) a poor understanding of genetic control of stress tolerance, (b) stress tolerance is often controlled by multiple genes, and (c) variation for stress tolerance usually exhibits a large environmental component or large environment by genotype interaction making direct selection for a physiological trait in a single environment difficult. Furthermore, tolerance at one developmental stage does not always confer tolerance at another stage. In addition, many methods proposed to monitor stress tolerance are based on the performance of individual cells, tissues, organs, or individual plants and do not provide a good indication of whole-plant response to stress either when grown in a spaced-plant nursery or in a competitive environment. Although competition has rarely been addressed as a selection criterion by plant breeders, both inter- and intraspecific competition have been shown to affect traits such as stable carbon isotope ratios and nutrient acquisition of plants (Williams et al., 1991;Caldwell et al., 1987). Similarly, Smith et al. ( 1992) demonstrated that grazing-tolerant alfalfa cultivars exhibited reduced persistence when grown in mixtures with tall fescue and grazed rather than when grazed in monocultures.
A. DEFOLIATION TOLERANCE Breeding perennial forage crops for improved defoliation tolerance is often a direct product of the selection that the breeder places on a cultivar before its release. Multiple defoliations within and between years by both man and grazers are commonly used in perennial forage breeding programs and are designed to allow only the most defoliation-tolerant genotypes to be released. Although studies have been conducted that compare interspecific variation for defoliation tolerance (Muir and Pitman, 1991), there have been few reports of breeders selecting for improved defoliation or grazing tolerance. Exceptions to this include breeding efforts in perennial turf grass species and efforts to develop grazing-tolerant alfalfa.
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Efforts to improve the grazing tolerance of alfalfa have resulted in the release of Alfagraze (Bouton ef al., 1991). Unlike earlier efforts to improve grazing tolerance, Alfagraze was developed under grazing. Two cycles of recurrent phenotypic selection were used in which a broad-based alfalfa population consisting of plants from 22 germ plasms or cultivars and from 1070 plant introductions were grazed for about I20 days in 3 years in cycle 1 and for 2 years in cycle 2. Subsequent evaluations of Alfagraze have indicated that it generally has many thick stems, intermediate decumbency, high herbage yield, many crown buds (Brummer and Bouton, 1991), maintains high levels of stubble carbohydrates, and high residual leaf areas (Brummer and Bouton, 1992) under frequent defoliation. Selection for rhizomes in perennial grasses and legumes is another trait that has been associated with improved persistence. Bouton ef al. (1989) reported that highly rhizomatous accessions of tall fescue survived better in competition with bermudagrass than did nonrhizomatous genotypes. Although Bermuda grass competition depressed rhizome production, tillering, and plant size across all genotypes, highly rhizomatous genotypes survived better than weakly or nonrhizomatous genotypes indicating that selection for rhizomatous cultivars could improve persistence in fescue. The presence of the endophyte did not affect rhizome production, making selection for rhizomes independent of endophyte infection status (De-Battista er af., 1990). In many tropical grass species, winter survival is associated with rhizome production. Bashaw ( 1980) selected Llano and Nueces buffelgrass (Cenchrus ciliaris L.) for rhizome production, which significantly improved the northern range of this species. The production of rhizomes, however, is influenced by soil fertility (M. A. Hussey and E. C. Bashaw, unpublished data) so that expression of rhizomes in buffelgrass and, thus, winter survival, depends on management. Similar results have been reported in nonrhizomatous warm-season grasses, where strategic use of defoliation and N fertilizer has been suggested as a method for the identification of winter-hardy genotypes of weeping lovegrass [Eragrosriscurvufa(Schrad.) Nees var. curvula Nees] (Voigt and DeWald, 1985). Recently, molecular markers associated with the rhizome trait have been reported in interspecific crosses between Sorghum bicolor x S. propinquurn (Paterson er af., 1995). The use of closely linked DNA markers and megabase DNA libraries provides the tools essential for map-based cloning of the genes regulating rhizome production.
B. DROUGHT TOLERANCE The use of controlled irrigation systems, such as line-source irrigation, rain-out shelters, and others, has enabled plant breeders and physiologists to identify genetic variation for crop water use and response to drought. Numerous reports have been published on the usefulness of carbon isotope discrimination (Cid) for pre-
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dicting water use in annual and perennial plants (Donovan and Ehleringer, 1994; Nageswara-Rao and Wright, 1994;Wright el al., 1994).Although heritabilities for water use efficiency (WUE) have generally been high, large environmental effects have been reported. Johnson et al. (1990) observed genetic variation in both crested wheatgrass and Altai wildrye [Leymus angustus (Trin.) Pilger] under both greenhouse and field conditions. Barker et al. (1989) also reported genetic variation for W E in crested wheatgrass, western wheatgrass [Pascopyrum smithii (Rydb.) A. Love], and intermediate wheatgrass [Thinopyrum intermedium (Host) Barkworth & D. R. Dewey], but noted that environmentalvariance was large. Asay and Johnson (1990) reported genetic variation for forage yield under water stress but observed that genetic variation for yield decreased as stress increased. In alfalfa, Johnson and Rumbaugh (1995) reported variation in Cid in some alfalfa populations but not in others. They concluded that, although variation in Cid was present, more diverse germ plasm would be needed to make substantial improvements in water use. Johnson and Tieszen ( I 994) measured Cid in a diverse set of alfalfa germ plasm originating from 13 countries. Total plant WUE was correlated with Cid only under drought stress; however, shoot WUE and Cid were correlated within well-watered and drought-stressed treatments. Reproducible variation for Cid was obtained in alfalfa germ plasm, suggesting that Cid is useful in evaluating alfalfa germ plasm for improved WUE. The use of seedling selection systems has indicated that genetic variation is available for seedling drought tolerance in lovegrass (Tischler et al., 1991). Hycrest crested wheatgrass was developed based on seedling drought tolerance (Asay et al., 1986). Because of its hybrid vigor and selection pressure in drought environments, Hycrest has had about 30% higher dry matter yields than Nordan in certain stress environments. Although selection for rooting depth in perennial forages has not been widely used, genetic variation for rooting depth has been reported in several species (Lehman and Engelke, 1993; Salaiz et al., 1991). In tall fescue, genotypes with thicker roots were reported to penetrate hardpans, have deeper rooting depths, and avoid water stress (Tolbert et al., 1990). Differences in rooting depth were noted between and within species with those having deeper rooting being more drought tolerant.
Most attempts to breed perennial forages under nutrient stress have focused on acid soils and genetic variation for growth in response to A1 (Edmeades et al., 1991; Lafever, 1981; Mackay et al., 1991;Mugwira andHague, 1993a,b,c,d). Genetic variation has been observed in many species including sorghum (Sorghum bicolor L. Moench) (Foy et al., 1993),perennial ryegrass (Wheeler et al., 1993a),
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alfalfa (Baligar et al., 1989; Bouton et al., 1986; Parrott and Bouton, 1990),lablab bean (Lablab purpureous L. Sweet) (Mugwira and Hague, 1993c,d), sesbania (Sesbania sesban L. Merr.) (Mugwira and Hague, 1993d), orchardgrass (Dactylis glomerata L.) (Wheeler et al., 1993b),tall fescue (Wheeler et al., 1993b),reed canarygrass (Phalaris arundinacea L.) (Oram et al., 1993), and others. Other research has focused on genetic variation for growth on acid soils (Alison and Hoveland, 1989; Bouton e f al., 1986)without consideration of specific nutrient stresses. Genetic variation for tolerance to high levels of Mg (Keisling et al., 1990), Cu (Plenderleith and Bell, 1990), or growth under low P (Mugwira and Hague, 1993a,b,c,d) has been identified. Although differences in genetic variation for ability to grow under nutrient stress have been identified, few cultivars have been developed with improved tolerance to acid soils or low nutrient status. This apparent lack of success by plant breeders is due to the complexity of nutrient uptake and the large interactions that exist between nutrients. Where tolerant cultivars have been developed, they have generally resulted from growing plants on nutrient-deficient soils and selecting those species with the best agronomic performance, rather than cultivars selected specifically for response to low nutrient stress.
D. SALINITYTOLERANCE Despite the existence of genetic variation for salinity tolerance, few salt-tolerant cultivars have been released (Noble and Rogers, 1993). Selection has been based primarily on agronomic characters such as yield and those traits that integrate physiological mechanisms responsible for tolerance. Genetic variation for salinity tolerance in perennial forage crops has been assessed during germination, seedling growth, and regrowth. Accessions of crested wheatgrass were identified with good germination and forage production under moderately saline (-0.6 MPa) conditions; however, consistent differences in salinity tolerance were not observed (Johnson, 1991). One of the most comprehensive studies of genetic variation of seedling salt tolerance compared populations of forage rape (Brassica napus L.), berseem clover, alfalfa, and red clover (Trifoliumpratense L.). Approximately 10,000 seedlings of each species were screened for growth at NaCl concentrations ranging from 200 to 250 mmol liter-'. A selection intensity of 1% was used with the plants exhibiting salinity tolerance being polycrossed and realized and narrow sense heritabilities were calculated. Realized heritabilities of 0.62, 0.34,0.31, and 0.57 were observed for forage rape, berseem, alfalfa, and red clover, respectively, suggesting that it should be possible to improve seedling response to saline conditions in these species (Ashraf et al., 1987). Fourteen half-sib families were randomly selected from an experimental alfal-
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fa population after two cycles of mass selection for improved forage growth at 80 mMNaCI. In separate experiments, the effect of salt stress was measured at germination (radicle growth at 7 days), during seedling growth (40 days after planting), and postharvest growth (forage yield at 67 and 95 days) under 0 and 80 mM NaCl. Although genetic correlations between seedling and regrowth yields were positive, it was concluded that selection for increased alfalfa forage yield in saline environments may not be optimum if conducted under saline conditions. Selection methods for improved salt tolerance should include several growth stages to develop alfalfa cultivars with improved yield under saline environments (Johnson et al., 1992). Recent advances in plant physiology and plant molecular biology have greatly expanded our understanding of physiological processes. Nevertheless, as with other agronomic crops, perennial forage cultivars must combine both quality and yield traits with tolerance to abiotic and biotic stresses. Except in rare circumstances, the advances made in understanding the physiology of abiotic stress tolerance will be used by plant breeders to develop selection techniques in which the genetic variation for specific traits can be quantified. Because of the wealth of interspecific and intergeneric variation that exists for tolerance to abiotic stresses, improvement efforts with perennial forages will continue to be targeted at the identification of tolerant species. Although genetic variation exists, most collections of perennial forages are small compared with cultivated species. Therefore, advancements in breeding for stress tolerance in perennial forages will likely follow the lead of cultivated crops, except for stress-tolerance traits that are uniquely important to perennial forages (e.g., winter hardiness, grazing tolerance, among others).
ACKNOWLEDGMENTS The authors thank Dr. David Briske, Texas A&M University, and Dr. Stan Wullschleger, Oak Ridge National Laboratory, for helpful critical reviews of an earlier version of the manuscript. M. Sanderson thanks the Texas Agricultural Experiment Station for granting a study leave at the Danish Institute of Plant and Soil Science, The Research Center Foulum, Denmark, to prepare this review. M. Sanderson also thanks Lise Molkier, Librarian at The Research Center Foulum for her help, and Villy J~rgensen and Dr. Christer Ohlsson, Department of Forage Crops and Potatoes, for the use of office space and facilities.
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