Integrative and Comparative Biology Advance Access originally published online on January 6, 2006
Integrative and Comparative Biology 2006 46(1):35-48; doi:10.1093/icb/icj006
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Nitrogen and carbon storage in alpine plants
*Department of Ecology and Evolutionary Biology, University of Colorado Boulder, Colorado 80309
Cooperative Institute for Research in Environmental Science, University of Colorado Boulder, Colorado 80309
Department of Biology, San Diego State University San Diego, California 92182
The Nature Conservancy of Nevada, Ely Nevada 89315
Correspondence: 1E-mail: Russell.Monson{at}colorado.edu
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 Synopsis IntroductionMaterials and methodsTheoretical considerationsCase study #1: Rhizome...Case study #2: Leaf...Conclusions and perspectives for...References |
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Alpine plants offer unique opportunities to study the processes and economics of nutrient storage. The short alpine growing season forces rapid completion of plant growth cycles, which in turn causes competition between vegetative and reproductive growth sinks during the early part of the growing season. Mobilization of stored nitrogen and carbon reserves facilitates competing sinks and permits successful completion of reproduction before the onset of winter stress. We discuss the theoretical framework for assessing the costs and benefits of nutrient storage in alpine plants in order to lay the foundation for interpretation of observations. A principal point that has emerged from past theoretical treatments is the distinction between reserve storage, defined as storage that occurs with a cost to growth, and resource accumulation, defined as storage that occurs when resource supply exceeds demand, and thus when there is no cost to growth. We then discuss two case studies, one already published and one not yet published, pertaining to the storage and utilization of nitrogen and carbon compounds in alpine plants from Niwot Ridge, Colorado. In the first case, we tested the hypothesis that the seasonal accumulation of amino acids in the rhizome of N-fertilized plants of Bistorta bistortoides provides an advantage to the plant by not imposing a cost to growth at the time of accumulation, but providing a benefit to growth when the accumulated N is remobilized. We show that, as predicted, there is no cost during N accumulation but, not as predicted, there is no benefit to future growth. In the presence of N accumulation, reliance on stored N for growth increases, but reliance on current-season, soil-derived N decreases; thus the utilization of available N in this species is a ‘zero sum’ process. Inherent meristematic constraints to growth cause negative feedback that limits the utilization of accumulated N and precludes long-term advantages to this form of storage. In the second case study, we discuss new results showing high concentrations of cyclic polyol (cyclitol) compounds in the leaves of many alpine species dominant in the dry fellfield habitat. In Artemisia scopulorum, cyclitols were induced as the growing season progressed, and reached highest concentrations during the dry, late-summer months. Leaf cyclitol concentrations were high in all four species of the Caryophyllaceae that we examined and appeared to be constitutive components of the leaf carbohydrate pool as concentrations were high through the entire growing season. We observed correlations among seedling abundance, seeding survivorship and the presence of high leaf cyclitol concentrations. We propose that the primary function of cyclitols in the leaves of alpine, fellfield herbs is to promote drought tolerance through osmotic protection, and enhance fitness by improving seedling survival. We considered the possibility that cyclitols also function as carbon storage compounds that are remobilized at the end of the growing season and used to support growth the following year. Our observations do not support this hypothesis in the Caryophyllaceae because the requirement for high constitutive concentrations year-after-year prevents long-term advantages of storage and remobilization. However, in A. scopulorum, remobilization of cyclitols following the end of the growing season may provide storage substrates that can be used for growth the following season. From our analysis we conclude that it is difficult to use current theory that is embedded in the economic concept of costs and benefits to interpret observed dynamics in nitrogen and carbon allocation. Future theoretical developments that move away from an abstract foundation embedded in cost-benefit tradeoffs and toward phenotypic integration of source-sink relationships will improve our ability to merge observations and theory.
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Introduction |
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SynopsisIntroductionMaterials and methodsTheoretical considerationsCase study #1: Rhizome...Case study #2: Leaf...Conclusions and perspectives for...References |
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Environmental and developmental influences on organismic function often cause asynchronies between the availability of critical resources and their utilization. In many species, a gap in resource supply and demand is filled by allocation to storage pools when supply is high, followed by remobilization to active metabolic pools as demand increases. The "benefit" of this strategy is clear in those cases where storage allows organisms to accommodate the gap, maintain function during periods of depleted resource and, ultimately, improve fitness. However, storage can also impose a "cost" if resources that are normally allocated to growth are channeled instead to inert storage pools. All organisms rely to some extent on resource storage. For example, resources are stored in eggs, seeds or specialized tissues during sexual and asexual reproduction. Considering only plants, resources are stored to (1) support growth following dormancy (Mooney and Billings, 1960
; Heilmeier et al., 1986; Cyr et al., 1990), (2) recover from herbivory or other natural catastrophes (Chiarello and Roughgarden, 1983; Lubbers and Lechowicz, 1989; Wyka, 1999; Scheidel and Bruelheide, 2004), and (3) accommodate pulses of resource surplus (Beck and Ziegler, 1989; Servaites et al., 1989; Li et al., 1992).
In general, reliance on storage should be correlated with the amount of asynchrony between resource supply and demand. Storage is predicted to be especially prevalent in species native to low resource habitats (Chapin, 1980, Chapin et al., 1990). As the value of a resource increases, the potential benefit of storing it when it is available, and using it when it is not available, is more likely to outweigh the cost of storage. Similarly, storage should be prevalent in habitats with short "windows of opportunity" for growth and reproduction (e.g., Schaeffer et al., 1983, Chiariello and Roughgarden, 1984; Jaeger and Monson, 1992). As the time available for growth and reproduction decreases, the demand for resources by these competing functions should increase, increasing the likelihood for asynchrony between supply and demand.
The concepts underlying resource storage have obvious analogies to the financial economics of human societies, and several theoretical treatments have been built on the concepts of economic tradeoff, including the costs and benefits of storage, the optimization of marginal gain, and the advantages of investments with multiple opportunities for return (e.g., Bloom et al., 1985; Chapin et al., 1990). To date, development of the theoretical foundations underlying resource storage have greatly eclipsed development of the observational foundations. In this paper, we briefly review past theoretical treatments. Then, we present two case studies of carbon and nitrogen allocation in alpine plants, one reviewed from the past literature and one not yet published, to illustrate the potential, or lack thereof, to integrate observations into the existing theoretical framework. Beyond these broader perspectives, the case studies provide more pointed lessons to the symposium at hand. Given the many contributions in this volume to the functional challenges that organisms face as they directly confront high-elevation extremes in the reduced partial pressures of gases, low ambient temperatures and high solar loads of visible and ultraviolet radiation, our paper provides breadth by pointing out one of the challenges organisms must indirectly face; the stresses imposed by lack of adequate time for successful completion of the seasonal growth and reproductive cycles.
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Materials and methods |
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SynopsisIntroductionMaterials and methodsTheoretical considerationsCase study #1: Rhizome...Case study #2: Leaf...Conclusions and perspectives for...References |
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The study site
Our research was conducted in the alpine tundra at 3290 meters in the Niwot Ridge Long Term Ecological Research (LTER) study site in the Front Range of the Rocky Mountains in Colorado. The studies of Bistorta bistortoides (discussed below in Case Study #1) were conducted in the moist meadow habitat of Niwot Ridge as previously described (Jaeger and Monson, 1992
; Lipson et al., 1996b). The growing season for plants in this habitat commences in early June after snow melt, with peak flower production occurring in mid-to-late July. Most leaves have senesced by early-to-mid October. The studies of leaf carbohydrate use (discussed below in Case Study #2) were conducted in the fellfield habitat of Niwot Ridge (see May and Webber, 1982). The fellfield is characterized as having a rocky substrate, mostly free of snow through the winter, high seasonal soil temperature fluctuations, and is the driest of the alpine communities in mid- to-late summer. This habitat is occupied by a large number of herbaceous dicots and monocots, most of which exhibit cushion and mat-forming growth habits. All fellfield data presented in this study were collected from three widely spaced sampling plots (separated by 100–150 m), on two separate knolls.
Analytical procedures
Sampling and analysis procedures for the studies of nitrogen storage in B. bistortoides have been discussed in detail in past papers (Jaeger and Monson, 1992; Jaeger et al., 1999; Lipson et al., 1996b) and will not be repeated here. Sampling for leaf carbohydrates in the fellfield plants occurred in the early morning, within no more than two hours after sunrise. Leaves were excised with a razor blade, placed in foil envelopes, and immediately frozen in liquid nitrogen. Samples were transported to the laboratory and stored in liquid nitrogen until lyophilized. Lyophilized samples were ground to a fine powder and stored at –20°C under a nitrogen atmosphere. Approximately 25 mg of ground leaf tissue was extracted in 1.5 cm3 of 80% ethanol. Samples were centrifuged (10,000 g, 15 min, 4°C), the supernatant was decanted, and pellets were resuspended in 80% ethanol. Ethanol extraction was repeated a total of five times, the fractions were pooled, and the ethanol was removed with a centrifugal evaporator. To remove pigments, dried fractions were resuspended in 1.5 cm3 of a 2:1 water:chloroform mixture. The aqueous phase was collected and soluble carbohydrates were measured by high-performance anion-exchange chromatography-PAD, essentially as described by Moore et al. (1997). Aldose and ketose sugars were separated isocratically (200 mM NaOH; 1 cm3/min) using a PA-1 column (Dionex, Sunnvale CA), sugar alcohols were separated isocratically (600 mM NaOH; 0.7 cm3/min) using an MA-1 column (Dionex, Sunnydale CA). Eluted carbohydrates were quantified with an ED-40 electrochemical detector with gold working electrode (Dionex, Sunnydale CA). Authentic standards were purchased commercially (Sigma). In this study, myo-inositol and associated isomers as well as the methylated inositols (e.g. ononitol and pinitol) were pooled and reported as ‘total cyclitols’.
Plant community analysis
Community composition was determined in July 2001 using a modification of the point intercept method as described by Barbour et al. (1987). We used 100 sampling points located 20 cm apart within each plot. Plant abundance was expressed using an absolute measurement of projected plant area (PPA; Jonasson, 1992) which is highly correlated with species biomass (Frank and McNaughton, 1990). In 1998, three plots of 1 m2 were created to monitor emergence and survival of seedlings. Seedling emergence censuses were conducted for three years (1998–2000) and survival censuses for four years (1998–2001) at approximately 2.5 week intervals from snowmelt (early to mid-June) through snowfall (early to mid-October); except in 2001, when survival censuses were conducted only at the beginning and end of the growing season. Dates of germination and mortality were recorded for each individual. Species identifications were made using photographs of lab-germinated seedlings.
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Theoretical considerations |
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SynopsisIntroductionMaterials and methodsTheoretical considerationsCase study #1: Rhizome...Case study #2: Leaf...Conclusions and perspectives for...References |
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Past theoretical treatments have led to a sometimes confusing, and contradictory series of terms and concepts. Bloom et al. (1985)
developed economic analogies to describe the costs of storage in both direct and indirect terms. Direct costs included the energy required to move compounds against concentration gradients and convert compounds among stored and non-stored forms, and the energy and biomass required for the construction of storage tissues. Indirect costs included the diversion of resources away from growth and into inert pools. Millard (1988) noted that definitions of storage up to that time were flawed by reliance on the concept that stored compounds are depleted when demand exceeds supply, and replenished when supply exceeds demand (e.g., Chapin and Shaver, 1988). This definition could not account for some observations, such as the storage of N by N-deficient plants (e.g., Millard et al., 1990; Monson et al., 1994). Millard (1988) proposed an alternative definition that separated storage into two classes: stored reserves that are mobilized from one tissue to support the growth of another and accumulated compounds that build up when resource supply exceeds the combined demands of growth and maintenance. By this definition, accumulated compounds are congruent with those assimilated during luxury uptake (e.g., Hommels et al., 1989); also, in this definition, we find an effort to move the discussion away from the previous economic analogies and toward the observed plant phenotype. Chapin et al. (1990) expanded Millard's treatment by focusing on the fact that reserve storage comes at a cost to growth, whereas accumulation does not. As stated by Chapin et al. (1990), "reserves compete with growth at the time the stores are produced." Monson et al. (1994) pointed out, however, that this definition does not account for those cases in which storage tissues are constructed at one point in time, but filled at another point in time. Heilmeier and Monson (1994) noted other deficiencies in the theoretical treatments to that time. For example, the theory failed to adequately capture the role of recycling in which compounds that fulfill a function other than storage at one time are mobilized at a later time to support growth. Soluble leaf protein, for example, is primarily composed of Rubisco and other photosynthetic enzymes, but can be remobilized to support the protein needs of new growth or reproduction as leaves senesce (Heilmeier et al., 1986; Millard et al., 1990). As another example, organic compatible solutes can be synthesized during drought or salt stress to protect enzyme and membrane function (Morgan, 1984), but broken down and used for other purposes when the drought ceases. Although Chapin et al. (1990) acknowledged these processes as examples of recycling, they did not put them into the dichotomous context of storage versus accumulation. Compounds such as these can potentially fill the same role as stored compounds in providing growth substrates, but have evolved under fundamentally different selection pressures than those compounds specialized for storage.
Heilmeier and Monson (1994) devised a new scheme for designating C and N allocation patterns in plants (Fig. 1). One of the new features of the scheme was inclusion of an explicit allocation category called interim deposition. Compounds that fall into this category include Rubisco and organic solutes that are used in primary and secondary metabolism at one point in time and mobilized as growth substrates at another point in time. Compounds in this category provide multiple opportunities for return on investment; a concept that allows for benefits in both the stored and remobilized states.
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Case study #1: Rhizome N storage in Bistorta bistortoides |
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SynopsisIntroductionMaterials and methodsTheoretical considerationsCase study #1: Rhizome...Case study #2: Leaf...Conclusions and perspectives for...References |
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In a series of studies in the mid-1990's we examined the nature and role of nitrogen storage in the rhizome of Bistorta bistortoides (Pursh), an alpine herb that occurs in virtually all alpine habitat types from wet meadow to fellfield (Jaeger and Monson, 1992
; Lipson et al., 1996b; Monson et al., 2001). In the Rocky Mountains, this species reproduces solely through sexual processes (Mooney, 1963), leaving its prominent rhizome to function as an organ of nutrient storage. The rhizome of B. bistortoides can persist for several decades, and some rhizomes on Niwot Ridge have been aged to at least 60–70 years using persistent leaf scars (D. Lipson, personal observation). There is typically a gradient of decreasing N concentration from the proximal to distal end of the rhizome, with the decrease averaging 25% (Lipson et al., 1996b). Most of the N in the rhizome is contained in the protein fraction, and appears to not be active in supporting seasonal shoot growth (Jaeger and Monson, 1992). A significant fraction, however, is present as free amino acids, which exhibit clear seasonal dynamics, increasing in the fall during leaf senescence and decreasing the following summer during new growth (Lipson et al., 1996b). We tested the hypothesis that the adaptive significance of rhizome N storage is to extend the growing season of B. bistortoides plants, allowing them to initiate growth earlier in the spring. Our observations revealed that, on average, there was no growth driven by stored N prior to the time of current-season soil N availability. In Figure 2, the data show a clear trend toward reductions in rhizome N content coincident in time with soil N uptake. The hypothesis that stored N allows an extension of the growing season, supporting growth before soil N becomes available, was not supported. Rather, stored N was used to support the high demand for resources that was encountered early in the growing season when leaves and inflorescences competed for substrates that could not be supplied in adequate portion by soil uptake alone. Our calculations showed that approximately 60% of the annual aboveground N requirement was met by mobilization from the rhizome.
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In B. bistortoides, the early-season supplementation with stored N provides a critical resource to complement the adaptive acceleration of phenological phases. In many plants from temperate ecosystems seasonal phenology is phased, and characterized by distinct, successive periods of vegetative and reproductive growth. This pattern is not evident in B. bistortoides, and other alpine species, in which vegetative and reproductive growth occur simultaneously. For example, plants of the leguminous alpine herb, Oxytropis sericera, exhibit a similar overlap of vegetative and reproductive growth and rely on stored carbohydrate reserves to support the high demands of such growth (Wyka, 1999
). Shading of leaves in O. sericera caused a reduction in stored reserves and reduced overall growth. A similar increase in the reliance on stored reserves has been predicted for desert annual species that have evolved similar accelerations in their seasonal phenology (Schaeffer et al., 1982).
We used an experimental approach to force plants of B. bistortoides into the mode of luxury N uptake in order to test the hypothesis that N accumulation provides long-term advantages to the plant. We hypothesized that accumulation carries the advantage of assimilating a nutrient at no cost to immediate growth, which can then be allocated toward the enhancement of later growth. In the words of Heilmeier and Monson (1994, p. 168): "Accumulated products will gain adaptive significance when a respite from the growth-limiting conditions causing ‘overflow’ leads to an increased demand for the previously accumulated resource." We fertilized plots in a moist meadow containing numerous B. bistortoides plants with NH4NO3 for a two-year period and observed subsequent dynamics in N uptake, storage, and growth for the two years of fertilization and the subsequent two years (Lipson et al., 1996b). After four years, fertilization resulted in an increase in the N content of rhizomes, but no change in seasonal aboveground growth (Fig. 3). Thus, there was no apparent cost to the storage of the additional N and, by definition, N had "accumulated." An accounting of seasonal N dynamics revealed that the accumulated N, however, did not enhance later growth, even four years after fertilization. In Figure 4, we have summarized the change in rhizome N and shoot N in non-fertilized and fertilized plants from the first date of growth to maximum biomass. In non-fertilized plants, approximately 56% of the N required to provision new aboveground growth came from stored N. In the fertilized plants, the provisioning of seasonal growth could be entirely accounted for by the early season decrease in rhizome N. This does not leave room for the utilization of N that might have been assimilated from soil uptake during the current season. Thus, there appear to be strong internal feedbacks, such that the accumulation of N causes the plant to increase its reliance on stored N while at the same time reducing its reliance on current-season uptake.
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Using the same fertilization experiment, we addressed the question as to how rhizomes of B. bistortoides accommodate the additional N provided during accumulation. Our measurements revealed that accumulated N is stored as free amino acids, with the N-rich amino acids, arginine,
-acetylornithine, and glutamine accounting for a combined 87% of the accumulated N (Lipson et al., 1996a; Lipson et al., 1996b). We asked the question whether rhizomes produce more cells or bigger cells to accommodate the accumulated N, or whether it is added to existing cells. Our measurements revealed that the volume of individual rhizome cells, the specific volume of the entire rhizome, and the mass per cell, did not increase in response to N accumulation (Table 1). Thus, there was no evidence for increased cell production or increased cell size as a means of accommodating accumulated N. Instead, more N was stored in existing cells. We also observed a decrease in the sucrose concentration of cells that accumulated N, presumably reflecting the increased cost of providing carbon skeletons as the accumulated N is assimilated into stored amino acid pools. We observed no significant decrease in starch concentration, the compound used for longer-term storage of carbohydrates. Thus, while we did not observe evidence of a cost to N accumulation at the level of the whole plant, we did observe evidence of a cost at the level of individual cells, at least with regard to sucrose depletion.
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We propose two possible mechanisms to explain the feedback between increased N accumulation and decreased reliance on current-season N uptake. First, it is possible that the feedback reflects developmental constraints to growth that are prominent in alpine herbs (see Körner and Menendez-Riedl, 1989
; Körner, 2003b). Bud preformation limits the capacity of many alpine herbs to adjust growth and take advantage of resource pulses (Diggle, 1997; Aydelotte and Diggle, 1997; Forbis and Diggle, 2001; Meloche and Diggle, 2001). Such constraints could preclude "respite from the growth-limiting conditions causing resource overflow," which was invoked by Heilmeier and Monson (1994). Second, it is possible that the depletion of sucrose limits the availability of the rhizome energy reserves required to drive soil N uptake (e.g., Raab and Terry, 1995). This would be a rather direct effect of excessive N uptake, essentially unbalancing the normal C/N status of the rhizome and draining active root cells of the carbohydrate resources required for further N uptake. We have illustrated the proposed nature of both of these feedbacks in Figure 5.
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Our conclusions about the lack of long-term advantage of N accumulation in B. bistortoides are dependent on the particular set of observations that we made, and may not be universally applicable. It is possible that in some years climatic conditions foster low rates of soil N mineralization and low potential for soil N uptake. In these years, the overflow situation may be relieved and N that had accumulated in previous years may be used to compensate for the current season's lack of N. We did not observe such conditions in the four years of our study. It is also possible that the fertilized condition we imposed on the plants causes phenotypic disruption and uncouples the processes and controls that would normally govern the remobilization and use of excessive resource stores. We certainly note the artificial nature of the fertilization treatments we applied. Finally, it is possible that we did not carry the experiment through to the point where the respite from the overflow situation was possible. Bud preformation in alpine plants can constrain growth for at least three years in advance (Diggle, 1997
). Our four-year experiment may not have been adequate to allow relaxation of the preformation constraint. If so, then this in itself is informative as to the extreme conditions placed on alpine plants with regard to their ability to capture resource pulses. Utilization of a resource overflow that is uncoupled from its initial capture by a stretch of time greater than four years would introduce considerable risk to the ultimate utilization of the resource. The probability of catastrophes (e.g., herbivory) increases with time of separation. Additionally, in the case of overflow due to the appearance of a resource pulse, an increase in the time of separation between uptake and use increases the probability for loss of the favorable climatic conditions that led to the pulse in the first place (assuming that resource pulses coincide with generally favorable conditions in other environmental variables). Clearly, the conservative growth habit of alpine plants has compromised the efficient utilization of stored resources, whether in the near- or long-term. Cost-benefit theories about the evolution of storage strategies in plants will likely have to be modified to deal with the long time frames and conservative growth habits of alpine plants. |
Case study #2: Leaf carbohydrates in fellfield herbs |
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SynopsisIntroductionMaterials and methodsTheoretical considerationsCase study #1: Rhizome...Case study #2: Leaf...Conclusions and perspectives for...References |
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A survey of carbohydrates in leaves of the twelve most dominant fellfield species on Niwot Ridge (see May and Webber, 1982
) revealed reliance on a broad range of compounds and evidence of strong phylogenetic affinity (Table 2). As an example of the diversity of carbohydrates observed, B. bistortoides relies almost entirely on glucose, fructose and sucrose, Castilleja puberula produces high concentrations of mannitol, and Trifolium nanum contains high concentrations of cyclitols. The greatest proportion of species in the fellfield belongs to the family Caryophyllaceae, and all species of Caryophyllaceae that we sampled had high concentrations of leaf cyclitols (Fig. 6). Additionally, the occurrence of raffinose, stachyose and the acyclic polyol, mannitol, differed among species. No sorbitol was detected in Acomastylis rossii, a member of the Rosaceae family, which has been reported to accumulate sorbitol (Waalart, 1980). In general, the content of individual carbohydrates was highly variable among species; however, when expressed as a percentage of the total soluble carbohydrate pool, two clear groupings emerged: (1) Trifolium and members of the Caryophyllaceae maintained a majority of the soluble carbohydrate pool as cyclitols; (2) The monocots, Carex and Luzula, exhibited little cyclitol accumulation and maintained a majority of soluble carbohydrate as sucrose. Species in the Carophyllaceae exhibited cyclitol concentrations between 40–50% of the total soluble pool, whereas sucrose, the carbohydrate most typically associated with leaf carbon relations, represented less than 25%. Previous observations have established that certain taxonomic groups (e.g., the Leguminosae and Myrtaceae, especially the genus Eucalyptus) contain high concentrations of specific cyclitols (see Phillips and Smith, 1974; Ford, 1984; Dittrich and Brandl, 1987; Merchant and Adams 2005); however, to our knowledge, our observations are the first to reveal high, taxonomic-specific concentrations in the Caryophyllaceae.
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Cyclitol compounds are found in all plants (Obendorf, 1997
; Loewus and Murthy, 2000). By definition, cyclitols are cycloalkanes containing one hydroxyl group on each of three or more ring atoms, and including a variety of methylated derivatives (IUPAC, 1992). In the formal sense, cyclitols are not oligosaccharides, although they share biochemical and physiological functions with compounds in the raffinose family oligosaccharides (RFOs) (Peterbauer and Richter, 2001). During stress, cyclitol compounds often accumulate in leaf cells, with especially high concentrations observed during salt stress (Gorham et al., 1984; Sacher and Staples, 1985; Klages et al., 1999; Arndt et al., 2004; Merchant and Adams 2005), water stress (Ford, 1984; Nguyen and Lamant, 1988; Paul and Cockburn, 1989; Keller and Ludlow, 1993; Wanek and Richter, 1997; Orthen et al., 2000; Streeter et al., 2001), and low temperature stress (Guo and Osterhuis, 1995; Orthen and Popp, 2000). Direct evidence of cyclitol accumulation as an osmotic protectant was obtained after transgenic overexpression of enzymes producing ononitol in leaves of tobacco plants, which resulted in enhanced photosynthesis during water and salt stress compared to wild-types (Vernon et al., 1993; Sheveleva et al., 1997).
In seeds, cyclitols are often mobilized during germination to generate energy and carbon substrates (Peterbauer and Richter, 2001; Karrer et al., 2004), a process that is consistent with their role as storage compounds. However, other cyclitols (e.g., the methylated cyclitols such as ononitol and pinitol which are common in legume leaves, and myo-inositol which is abundant in most leaves) turn over slowly (Smith and Philips, 1982; Paul and Cockburn, 1989; Wanek and Richter, 1997; Klages et al., 2004), suggesting a role more specialized in metabolic protection than carbon storage. The role of cyclitols as osmotic protectants that enhance function during drought may be particularly important to the survival of seedlings. Gorecki et al. (1997) found that cyclitols accumulated during germination in yellow lupine seeds, and enhanced germination after desiccation treatments. Gutterman (2001) found that seedling survival was correlated with cyclitol concentration in the desert annual plant, Schismus arabicus. Recent studies in Quercus robur trees indicated the possible dual role of cyclitols in storage and protection; repeated defoliation caused a reduction in soluble sugars and cyclitols in bark tissues and, at the same time, reduced the ability of these tissues to acclimatize to frost (Thomas et al., 2004).
Our observations of seasonal dynamics in leaf cyclitol concentration revealed three distinct types of response (Fig. 7). In species such as B. bistortoides, Acomastylis rossii and Carex rupestris, cyclitol concentrations were relatively low across the entire season. In species such as Artemisia scopulorum, cyclitol concentrations were relatively high and increased as the growing season increased, reaching a maximum late in the season when drought stress is prevalent (see Jaeger et al., 1999). In species of the Caryophyllaceae, cyclitol concentrations were high through the entire season; there was no evidence of induction as the season progressed. In all species, we found high concentrations of cyclitols in over-wintering leaf primordia that were produced at the end of the growing season. This late-season retention is likely associated with cryoprotection or wintertime desiccation tolerance; the snowpack is thin to non-existent in the fellfield, offering these plants little protection from thermal extremes during the winter.
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We hypothesized that the high concentrations of cyclitols in leaves enhance survival in the dry, fellfield habitat. Careful observations of seedling demography revealed that seedling abundance was not correlated with the abundance of adult plants (Fig. 8). The species with highest seedling abundance were those in the family Caryophyllaceae and Trifolium nanum, the species with the highest concentrations of leaf cyclitols. Seedling survival over three successive years was also highest among species with the highest concentrations of leaf cyclitols (Fig. 9). All seedlings that survived all three growing seasons were from species that exhibit high leaf cyclitol concentrations. Surprisingly, Arenaria fendleri which belongs to the Caryophyllaceae and exhibits high concentrations of cyclitols, did not exhibit higher-than-normal rates of seedling survival. However, plants of A. fendleri are rare in the fellfield compared to other members of the Caryophyllaceae, and seedlings occur infrequently (Fig. 8). There are clearly factors other than the presence or absence of cyclitols that limit the occurrence of this species in this habitat.
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If cyclitols have a role in enhancing seedling survivorship in the fellfield habitat, it will be important to demonstrate that cyclitol concentrations are high in seedlings, as well as adults. Due to the minimal biomass of alpine seedlings (0.2–1.0 mg dry mass), determination of carbohydrate composition at this life-stage remains a technical challenge. However, in a pooled analysis of 12 field-collected Silene acaulis seedlings, cyclitols comprised approximately 48% of the soluble carbohydrate pool (data not shown), consistent with their concentration in adult tissues, and consistent with their proposed protective role in seedlings.
The accumulation of cyclitols in fellfield Caryophyllaceae and the potential for cyclitols to enhance seedling survival in dry and cold conditions suggests the possibility that cyclitols are an adaptation to extreme environments. Members of the Caryophyllaceae are found in cold climates throughout the globe and two Caryophyll species hold the records for the southernmost dicot species (Colobanthus quitensis, Antarctica; Ruhland and Day, 2001), and the highest-elevation vascular plant species (Arenaria bryophylla, at 6,180 m in the Himalayas; Polunin and Stainton, 1997). These observations suggest that further study of the functional significance of cyclitols and their distribution throughout this family might shed light on an important physiological adaptation that has allowed vascular plants to colonize and survive in extremely cold regions.
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Conclusions and perspectives for further development |
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SynopsisIntroductionMaterials and methodsTheoretical considerationsCase study #1: Rhizome...Case study #2: Leaf...Conclusions and perspectives for...References |
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It was difficult to merge the observations described in our two case studies with existing theory. We were not able to clearly classify the unfertilized, seasonal buildup of N in the rhizome of B. bistortoides as "reserve storage" or "accumulation." On theoretical grounds, the partitioning of resources to a heterotrophic storage organ, such as that in B. bistortoides, should carry a cost to growth (e.g., Monson et al., 1994
). The theory is less clear on when and whether this cost is realized, given the multiple influences on growth that exist in situ. In several recent papers, Körner and co-workers (e.g., Hoch et al., 2002; Hoch and Körner, 2003; Körner, 2003a) have argued that high-elevation plants are not limited by resource availability, and by inference, the seasonal storage of resources does not limit growth. If true, then the actual costs to growth of allocating carbon and nitrogen to the construction and filling of storage tissues may be lower than predicted by current theory. We were only able to state with certainty that the storage of nitrogen reflected accumulation, rather than reserve storage, in the fertilized plants; an artificial condition that may not be relevant at all to the native condition.
One critical issue that has seldom been confronted, and one that we struggled with in our own studies, concerns the basis against which one evaluates the costs of storage. As described above for existing theory, the term "cost" implies that some increment of potential growth is lost because resources are allocated to inert pools. Given this framework, the question can be posed: what is the original growth rate that we should use as the basis for the lost increment in potential growth? Depending on which frame of reference is used, we were able to come to completely different conclusions about the costs of storage in B. bistortoides. If, for example, one uses the average growth rate of the plant per unit nitrogen, and calculates the potential for growth if the stored N were allocated to leaves rather than storage pools (sensu Monson et al. 1994), then limitations not related to resource supply (e.g., bud preformation) are ignored and the cost will be overestimated. With this approach, we would conclude that the cost of seasonal N storage in B. bistortoides is relatively high. If, instead, we recognize that the seasonal increase in rhizome N occurs during the late summer or early autumn, when growth has ceased (Fig. 2), we could conclude that seasonal N storage in B. bistortoides reflects accumulation, with no cost to growth. If, as another alternative, we recognize that calculated costs should include the production of storage tissues early in the plant's life cycle, before storage pools are filled (e.g., Monson et al., 1994; Heilmeier and Monson, 1994), then the growth rate during early phases of the growth cycle would be most relevant in calculating the cost. Given the long life span of B. bistortoides, costs early in life will be amortized over many years, resulting in rather high overall costs to storage. Finally, storage can be viewed in ultimate terms; that is, we can take the perspective that the evolution of a fixed storage strategy has acted as a selective filter that has constrained the evolutionary potential of the plant and forced inherent limits on the overall growth rate. With this perspective, the relevant growth rate for calculating cost would be that for a theoretical conspecific plant that does not rely on storage. Once again, given the potential for this type of cost to be amortized over the life of the plant, the calculated costs could potentially be high. Clearly, there is room for modifications to the theory of storage to reconcile these frames of reference and articulate them into a rational means for estimating costs.
With regard to the buildup of carbon compounds in the leaves of fellfield herbs, we were similarly challenged in our effort to merge theory and observations. We were not able to determine whether the constitutive buildup of cyclitols reflects "interim deposition" as defined in past theory (Heilmeier and Monson, 1994). While our studies demonstrate that cyclitols reach high concentrations in the leaves of some alpine plants, which is consistent with a role in carbon storage, and are correlated with improved seedling survival in dry alpine habitats, which is consistent with a role in protection from water stress, we were not able to show that the cyclitols are metabolized and used to support future growth, which would be required of any compounds having a role in interim deposition. To date, catabolic metabolism capable of mobilizing leaf cyclitol compounds has not been described, and it is generally believed that these compounds turn over slowly (e.g., Wanek and Richter, 1997). It is also believed that time-dependent changes in the concentration of cyclitols in leaf tissues is more likely due to translocation and storage as intact molecules elsewhere in the plant, than breakdown and remobilization in a different form (Popp et al. 1997). Furthermore, given the constitutive nature of cyclitols in the Caryophyllaceae, even if turnover and remobilization were possible, there would be no advantage to the overall carbon economy of the plant; the required maintenance of constitutive levels would prevent opportunistic mobilization of cyclitols as a source of growth substrates.
Many of the difficulties we encountered in integrating theory with observations were due to limitations in the theory itself; particularly with regard to the theoretical underpinnings of possible tradeoffs between growth and reproduction, a critical issue in the case of the accelerated reproductive cycles observed in alpine herbs. The traditional interpretation of the relationship between growth and reproduction is that constraints to the latter can be relieved by the former; i.e., more growth leads to a larger plant which in turn leads to higher fecundity. This requires a phased annual growth cycle in which a ‘resource capacitance’ is established through vegetative growth early in the season, followed by a switch later in the season when the capacitance is expended and resources are channeled from vegetative to reproductive tissues. The phased growth strategy also permits flexibility in the use of growth meristems; meristems can be dedicated to vegetative growth early in the cycle, then switched to reproductive growth later in the cycle. Acceleration of the reproductive cycle forces overlap in the development and expenditure of resource capacitance, and forces meristems for vegetative and reproductive growth to be activated coincidently in the early portion of the growth cycle and, in many cases, preformed the previous year. This imposes two costs to growth; one due to the fact that substrate normally targeted for growth must now be channeled to reproduction and one due to the fact that preformed meristems require time for development, provisioning and hardening, forcing growth to become more deterministic. These costs are not independent of each other. The deterministic tendencies of preformation will constrain growth in a manner that reduces the cost of substrate deficiency and limits the benefit of future growth from remobilized stores. To date, preformation, and its potential to uncouple storage from the costs and benefits traditionally assigned to it, has not been included in theoretical treatments.
Given the special circumstances of the case for alpine herbs, we propose a modified and simplified version of the model originally presented in Figure 1 (see Fig. 10). It is hoped that this form of the model will better reflect the theoretical needs for evaluating the special roles of storage in alpine plants. We propose that the principal relations that need clarification are (1) the degree to which the size of the storage pools, tendency toward bud preformation and capacity for current-year resource uptake influence growth and reproduction, (2) the degree to which growth and reproduction compete for growth substrates, and (3) the potential feedbacks from storage and accumulation to preformation and current-year resource uptake. Improvement of the models in these areas will require the cost-benefit components of current theories to be phrased in less abstract terms, and integrated with the known physiological processes that govern growth and allocation; the models must become more ‘phenotype relevant’. There are clear challenges here as our knowledge of the processes underlying growth and regulation is limited. Nonetheless, it is hoped that by simply recognizing the need for this type of framework, we can focus future studies toward a more targeted articulation of the connections between resource acquisition, physiological sources and sinks, growth and fecundity as components of fitness in alpine plants.
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From the symposium "Adaptations to Life at High Elevation" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4–8, 2005, at San Diego, California.
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SynopsisIntroductionMaterials and methodsTheoretical considerationsCase study #1: Rhizome...Case study #2: Leaf...Conclusions and perspectives for...References |
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