Summary

I. INTRODUCTION – A HYPOTHESIS 374

II. EFFECTS OF CALCIUM ON PHYSIOLOGICAL PROCESSES 376

1. The chemical uniqueness of calcium 376

(a) Cytotoxicity 376

(b) Binding properties 376

(c) Stimulation/displacement potential 376

2. Calcium signaling and plant responses to environmental stress 377

(a) Control principles 377

(b) Carbohydrate metabolism 378

(c) Synthesis and function of membranes and cell walls 379

(d) Disease resistance and wound repair 380

(e) Cold tolerance 381

(f) Stomatal regulation 382

III. CALCIUM UPTAKE AND DISTRIBUTION AT THE WHOLE-PLANT LEVEL 382

1. Uptake at the root-soil interface 382

2. Transport and exchange in stems 384

3. Exchange of calcium by foliage 385

IV. ECOSYSTEM PROCESSES AND CALCIUM SUPPLY 387

1. Plant succession and soil acidification 387

2. Plant adaptations to nutrient deficiency 389

(a) Morphological adaptations 389

(b) Physiological adaptations 390

V. PLANT AND ECOSYSTEM RESPONSES TO HUMAN ALTERATIONS IN CALCIUM SUPPLY 391

1. Increased atmospheric inputs of acidity 391

(a) Reductions in soil cation pools 392

(b) Inhibition of calcium uptake and effects on root function 393

(c) Increased leaching of calcium from foliage 395

(d) Physiological indicators of altered forest function 397

(e) Wood chemistry, structure and function 402

2. Forest management 404

(a) Harvesting effects on nutrient supply 404

(b) Managing forest nutrient supply 405

VI. CONCLUSION 407

1. Whole-tree perspectives 407

2. Ecosystem perspectives 408

VII. EVALUATION OF THE HYPOTHESIS 410

Acknowledgements 411

References 411

Summary

Calcium occupies a unique position among plant nutrients both chemically and functionally. Its chemical properties allow it to exist in a wide range of binding states and to serve in both structural and messenger roles. Despite its importance in many plant processes, Ca mobility is low, making Ca uptake and distribution rate a limiting process for many key plant functions. Ca plays an essential role in regulating many physiological processes that influence both growth and responses to environmental stresses. Included among these are: water and solute movement, influenced through effects on membrane structure and stomatal function; cell division and cell wall synthesis; direct or signaling roles in systems involved in plant defense and repair of damage from biotic and abiotic stress; rates of respiratory metabolism and translocation; and structural chemistry and function of woody support tissues. Forest trees, because of their size and age capacity, have been examined for evidence of limitations imposed by the timing and level of Ca supply. Examination of Ca physiology and biogeochemical cycling for forested systems reveals many indications that Ca supply places important limitations on forest structure and function. These limitations are likely to be most significant with older trees, later successional stages, high levels of soil acidity and/or high canopy Ca leaching losses, or under conditions where plant competition is high or transpiration is limited by high humidity or low soil moisture. Evidence of structural and physiological adaptations of forests to limited Ca supply; indicators of system dysfunction at many levels under reduced Ca supply; and the positive responses of diverse indicators of forest vitality in liming experiments indicate that Ca is more important to forest function and structure than has generally been recognized. Lack of recognition of Ca limitations is due in part to that fact some important plant functions are controlled by changes in very small physiologically active pools within the cytoplasm, and whole-leaf Ca levels may not reflect these limitations. An additional aspect is the fact that Ca availability has declined significantly for many forests in just the past few decades. Additional research on the role of Ca supply in resistance of forests to disease, changes in structural integrity of woody tissues, restrictions on rooting patterns and function, and uptake of other nutrients, notably N, is needed. Increased understanding of the physiological ecology of Ca supply can be anticipated to provide important insights that will aid in future protection and management of both natural and commercial forest systems.

Frictional resistance to a penetrating body can account for more than 80% of the total resistance to penetration of soil. We measured the frictional resistance between growing root caps of maize and pea and ground and smooth glass surfaces, which was linearly correlated to load, allowing calculation of the coefficient of kinetic friction and adhesion. Coefficients of kinetic friction between the root caps and the ground and smooth glass surfaces were approximately 0.04 and 0.02, respectively, the first measurements of the frictional properties of root tips at rates approaching those of root elongation, and an order of magnitude smaller than those previously reported. Results suggest that roots are well designed for penetrating soil, and encounter only small frictional resistance on the root cap. These data provide important parameters for modelling soil stresses and deformation around growing root tips.

One alkaline invertase and two acid invertase activities were detected in the shoots of etiolated rice (Oryza sativa) seedlings. The alkaline invertase (AIT) was purified to homogeneity through steps of ammonium sulphate fractionation, concanavalin A-Sepharose affinity chromatography (non-retained), DEAE-Sephacel chromatography and preparative electrophoresis. The pH optimum of AIT was 7.0 and the molecular mass, determined by gel filtration, was 240 kDa. It is apparently a homotetrameric enzyme (subunit molecular mass 60 kDa). The isoelectric point was 4.4 by isoelectric focusing. The best substrate of the enzyme was sucrose, with a Km of 2.53 mM. The enzyme also hydrolysed raffinose, but not maltose or lactose, so it is a β-D-fructofuranosidase. It gave negative glycoprotein staining. Of the hydrolysis products, fructose was a competitive inhibitor and glucose was a non-competitive inhibitor. Treatment with an alkaline phosphatase could activate AIT, whereas other proteins such as BSA, concanavalin A and urease had no effect on the enzyme activity. The enzyme activity was inhibited by Tris, thiol reagents and heavy metal ions.

Spatial distribution of cell turgor pressure, cell osmotic pressure and relative elemental growth rate were measured in growing tall fescue leaves (Festuca arundinacea). Cell turgor pressure (measured with a pressure probe) was c. 0.55 MPa in expanding cells but increased steeply (+0.3 MPa) in cells where elongation had stopped. However, cell osmotic pressure (measured with a picolitre osmometer) was almost constant at 0.85 MPa throughout the leaf. The water potential difference between the growth zone and the mature zone (0.3 MPa) was interpreted as a growth-induced water potential gradient. This and further implications for the mechanism of growth control are discussed.

The influence of variation in the concentration of dissolved inorganic carbon (DIC) in the form of CO2 and HCO3− in the root media on the C and N metabolism of Lycopersicon esculentum cv. F144 was investigated under both saline and non-saline conditions. Tomato seedlings were grown in hydroponic culture (pH 6.5) with or without NaCl, and the root solution was aerated with either ambient CO2 (360 μmol mol−1) or CO2-enriched air (5000 μmol mol−1). Nitrate uptake and root tissue NO3− concentrations were increased slightly by elevated rhizosphere DIC concentrations in both control and salinity-treated plants. This was associated with 46% higher nitrate reductase activity in the roots of control plants supplied with elevated DIC than in those supplied with ambient DIC. The activity of phosphoenolpyruvate carboxylase (PEPc) in vitro in control and salinity-treated plants was unaffected by the supply of elevated rhizosphere DIC concentrations. However, PEPc activity in vitro was considerably higher than the rates of PEPc activity in vivo reported previously, indicating that PEPc activity was not in itself a limitation on the provision of anaplerotic C. Therefore elevated DIC concentration in the rhizosphere stimulated the uptake of NO3− and provided alternative C skeletons for the assimilation of the NH4+ resulting from NO3− reduction into amino acids within the roots. Salinity stimulated root glutamine synthetase (GS) activity up to double that in control plants. Furthermore, elevated DIC caused an increase in leaf and root GS activity of control plants while inhibiting GS activity in the roots of salinity-treated plants. Glutamine[ratio ]2- oxoglutarate aminotransferase (GOGAT) activity of salinity-treated plants was doubled by elevated rhizosphere DIC concentrations. These changes in GS and GOGAT activity must reflect changes in amino acid synthesis. Under saline conditions the xylem transport of NO3− is partly blocked and a larger root assimilation develops, requiring not only the transamination of 2-oxoglutarate to glutamate but also that of oxaloacetate to aspartate and the transamidation of aspartate to asparagine.

Nitrogen (N) allocated to leaf growth in forage grasses and legumes following severe defoliation is predominately mobilized from the remaining root and leaf sheath tissues, since both N uptake from the soil and N2 fixation are severely down-regulated for several days. The hypothesis that a low N reserve status at the time of defoliation limits N remobilization and leaf regrowth was tested with contrasting cultivars of Lolium perenne (cvs Aberelan and Cariad) in flowing solution culture. Plants were grown under ‘high’ or ‘low’ (uptake of N decreased by 50%) regimes of N supply for 10 d before a single severe defoliation. Labelling with 15N was used to assess the importance of N reserves, including putative vegetative storage proteins, relative to N translocated from concurrent uptake, as a source of leaf N during regrowth. Leaf regrowth, N uptake and N mobilization were all affected by previous N supply. Low plant N status at the time of defoliation increased regrowth dry weight of ‘Aberelan’ by 10% and translocation of N absorbed from the medium by 23%, while mobilization of N reserves was decreased by 56%. On the contrary, regrowth dry weight of ‘Cariad’ was decreased by 23%, and translocation of N absorbed by 21% in low plant N status, compared with high plant N status. Concentrations of soluble protein in roots and remaining leaf sheaths decreased after defoliation in plants only under optimal N supply. Analysis of soluble proteins in sheath material by SDS–PAGE suggested that three polypeptides (55, 36.6 and 24 kDa) might function as vegetative storage proteins, although they were of low abundance in plants, subjected to monthly harvests, grown in controlled conditions and in the field. The apparent antagonism between uptake of NH4+ or NO3− by roots and mobilization of N reserves is discussed together with evidence for functional vegetative storage proteins in L. perenne.

Applying silicon in the form of metasilicic acid (H4SiO3) or silicic acid (H4SiO3) to Bradyrhizobium-infected, hydroponically grown cowpea seedlings resulted in a significant (P[les ]0.05) increase in the number of nodules, nodule dry matter, and nitrogen fixed on a per plant basis. Total dry matter of plants increased with silicon supply, and the differences were significant (P[les ]0.05) at the higher silicon concentrations. Cowpea plants cultured in sand were also assessed for their response to silicic acid. Nodule number and nodule mass increased with silicon supply to sand cultured plants, though nitrogen fixation was unaltered. Although silicon is not essential for growth of cowpea, it is important for nodule formation and nodule functioning in hydroponically grown plants. Consequently, data collected and conclusions drawn from earlier glasshouse experiments, which have excluded silicon from nutrient solutions, may be flawed. Future studies on nodulation and nitrogen fixation using legumes in liquid culture must therefore include silicon as a nutrient element.

Canopies of heterophyllous trees expand by production of long shoots. We have previously shown in mountain birch (Betula pubescens ssp. czerepanovii) that damage to internode leaves within long shoots does not impede shoot growth, indicating that long-shoot elongation occurs by means of external resources. To study to what extent leaves other than true long-shoot leaves are necessary for the normal growth of mountain birch long shoots, we simulated herbivore damage to the two basal leaves of shoots (which flush simultaneously with short-shoot leaves) and the short-shoot leaves nearest to the long shoot within the branch. Damage to the two basal long-shoot leaves significantly reduced long-shoot growth. Additional damage to short-shoot leaves, situated proximally to the long shoot, did not retard long-shoot growth any more than damage to basal leaves alone. To determine the extent to which short-shoot leaves within a large branch are responsible for the pooled long-shoot production of the branch, we clipped differing proportions of short-shoot leaves from such branches. We found small but significant reduction in the pooled length of the long shoots of the branch, presumably indicating a limited role in long-shoot elongation of current photosynthates within the branch. Our experiments indicate that long shoots are not independent modular units in their carbon economy.

Grasses and forbs are often classified into separate functional types, although systematic differences between the types have only been verified for a few functional traits. Since leaf longevity has been shown to be a key trait linking plant ecophysiology, whole-plant growth and ecosystem resource cycling, we compared the leaf longevity of 14 species to determine if there were consistent differences between grasses and forbs or other functional classifications, such as persistence of leaves into winter. Leaf longevity was assessed in 6-yr-old monoculture plots in central North America by tagging and sequentially monitoring the phenological states of whole forb leaves and sections of grass leaves. This new approach enables a calculation of leaf longevity unbiased by the manner in which grass leaves grow and provides a more accurate comparison between grasses and forbs. Lupinus perennis had the shortest leaf longevity (4 wk) and Koeleria cristata, Poa pratensis, and Solidago rigida the longest (13–14 wk). Average leaf longevity for the 14 species was c. 9 wk, with no significant differences between grasses and forbs nor between current alternative functional classifications.

Eupatorium makinoi plants with or without geminivirus infection were grown in shading frames with 70, 15 and 5.5% sunlight. Growth characteristics of these plants in the early vegetative phase were compared by means of growth analysis. We also measured leaf photosynthetic gas exchange rates and examined relationships between leaf photosynthesis and whole-plant growth. Relative growth rate (RGR=(1/W)×(dW/dt), where W is plant dry mass) of virus-infected plants was lower than that of uninfected plants under all three light conditions. The reduction of RGR by infection was increased with irradiance. The net assimilation rate (NAR=(1/A)×(dW/dt), where A is total leaf area of the plant) was also reduced both by infection and shading. NARs that were estimated from light-response curves of leaf photosynthesis, in situ measurements of irradiance, and respiration rates of leaves, stems and below-ground parts, agreed very well with the values obtained by conventional growth analysis techniques. Decreases in the estimated NAR value from infection and shading were mostly explained by the decreases in leaf photosynthesis. These results clearly showed that lowered RGR in virus-infected plants was attributed mainly to impaired photosynthesis in virus-infected leaves.

Colonization of roots by arbuscular mycorrhizal (AM) fungi in several annual crops in two consecutive seasons was compared with, in the second season, the density of fungal propagules in the soil with the use of a bioassay. Root density decreased down the soil profile in both years in all crops, and a high proportion of roots were mycorrhizal throughout the profile. AM colonization decreased down the profile in cotton and lablab in the second season only. The bioassay indicated that most propagules of AM fungi in soils under cotton were located near the surface, with virtually no propagules at 1 m. The absence of propagules at depth indicates a lack of mycelium deep in the soil, and suggests that mycorrhizas are primarily initiated in the surface soil and that the fungi colonize the root system mostly through secondary spread down the profile. The use of AM colonization in the field as an indicator of propagule density and symbiotic function should be qualified by an understanding of the depth in the soil from which roots were extracted.

Functional morphological patterns in root apices of tomato (Lycopersicon esculentum) dependent on growth, ageing and infection by the arbuscular mycorrhizal (AM) fungus Glomus mosseae and/or by the soilborne pathogenic fungus Phytophthora nicotianae var parasitica (P. parasitica) were studied. Uninfected root apices were characterized by closed, tri-layered meristems with nonreticulate nuclei; however, some apices of each treatment lost their meristematic nature, stopped growing and differentiated, becoming ‘parenchymatized’. The pathogenic fungus reduced the apex diameter and the number of mitotically active and viable apices inducing plasmolysis, cell and nucleus degeneration, and necrosis. The AM fungus, on the other hand, produced an increase in apex size and reduced the percentage of necrosis both in uninfected roots and in roots infected by P. parasitica. Thus, the AM fungus protected the apices from the pathogenic infection, allowing normal root growth. Furthermore, larger apices, which produce thicker roots, might indirectly contribute to plant protection. Increased volumes of colonizable tissues favour the spreading of the symbiont, and P. parasitica hyphae are always excluded from arbuscule-containing cells.

Arbuscular mycorrhizal (AM) symbioses are known to play a role in increased resistance of plants against soilborne pathogens. Mechanisms involved in this phenomenon are not yet well understood. This work investigates possible roles of endoproteolytic activities in bioprotection of Pisum sativum roots by Glomus mosseae against Aphanomyces euteiches. First, it is demonstrated that bioprotection occurs only in pre-mycorrhizal plants. Second, endoproteolytic activities were analysed qualitatively and quantitatively during AM symbiosis, in plants infected with either zoospores or mycelium of A. euteiches, and in mycorrhizal plants infected with the pathogen. In mycorrhizal symbiosis a progressive increase in endoproteolytic activities was observed following root colonization by G. mosseae. By contrast, in roots inoculated with A. euteiches, a drastic increase in endoproteolytic activities was observed which was correlated with the amount of pathogen occurring in roots. Qualitative differences were seen among the endoproteolytic activities detected in roots inoculated with zoospores or mycelium. The constitutive as well as mycorrhizal and pathogen-induced activities were further characterized as ‘trypsin-like’ serine endoproteases. Interestingly, in a situation of bioprotection, only low levels of the activities normally associated with the infection by A. euteiches were detected, suggesting that the synthesis of these proteins is directly linked to the growth or virulence of the pathogen.

A glasshouse experiment was done to assess the development and phosphate metabolism of mycorrhizas formed by species of arbuscular mycorrhizal fungi (AMF) from two different genera, Gigaspora and Glomus on Desmodium ovalifolium plants at three concentrations of a phosphate source. The addition of phosphate (0–100 mg P kg−1) had no effect on the alkaline phosphatase activity, stained histochemically, in the intra-radical mycelium of Gigaspora rosea (BEG111), but decreased that of Glomus manihotis (BEG112) over a 10-wk period. The alkaline phosphatase activity of the extra-radical mycelium was unaffected by increasing phosphate addition (0–100 mg P kg−1) in both species of AMF over a 10-wk period. The extra-radical mycelium of Gi. rosea (BEG111) accumulated polyphosphate, determined by staining with 4′,6-diamidino-2-phenylindole, whereas polyphosphate was not detected in the extra-radical mycelium of G. manihotis (BEG112). This work indicates differences in the mechanisms of phosphate metabolism in the mycelium of AMF from different genera on a tropical host. This might be determined by the life-cycle strategies of these fungi, in particular the formation of auxiliary cells in Gigaspora. The possibility of a negative-feedback mechanism between alkaline phosphatase and polyphosphate in the extra-radical mycelium of Gi. rosea (BEG111) and the role of polyphosphate in the symbiosis are discussed.

A factorial analysis was conducted to investigate the effects of different levels of photosynthetic photon flux (PPF) and CO2 concentration on the interactions between the vesicular–arbuscular endomycorrhizal fungus Glomus intraradices and potato plantlets (Solanum tuberosum) cultured in an in vitro tripartite system. We observed that CO2 enrichment from 350 to 10000 ppm stimulated root colonization by the fungus, and that this stimulation was more pronounced under high PPF (300 μmol m−2 s−1) than low PPF (60 μmol m−2 s−1). Consistent with these observations, the effects of G. intraradices on dry matter production in potato plantlets were strongly dependent on the CO2 and PPF levels during cultivation. There was no significant effect of the mycorrhizal fungus on dry matter production at 350 ppm of CO2. However, under the high CO2 concentration, mycorrhiza had opposite effects on dry matter production depending on the PPF: a decrease (−21%) and a stimulation (+25%) of dry matter production after 2 wk of growth under low and high PPF, respectively, were observed in presence of G. intraradices relative to plantlets grown in its absence. Furthermore, in mycorrhizal plantlets grown under high levels of both PPF and CO2, the chlorophyll and carotenoid contents as well as the quantum yields of photosynthetic electron transport and the photochemical quenching qP of the chlorophyll-a fluorescence measured near the PPF during growth were all higher than in non-infected plantlets. Our results therefore indicate that mycorrhizal G. intraradices can alleviate the down regulation of photosynthesis related to sink limitation, and its effect on dry matter production is strongly dependent on the levels of CO2 and PPF during growth which determine the balance between the photosynthetic carbon uptake by the plantlets and the carbon cost by the fungus.

The effect of arbuscular mycorrhizas on fructan accumulation was studied in barley (Hordeum vulgare) infected with Glomus mosseae. Treatments with and without fertilizer were included in order to distinguish between mere fertilizer effects and the effects of the symbiosis, and plants were harvested at two different time points, 35 and 50 d after planting. Fructan was the major storage carboyhdrate in both leaves and roots. The amounts of fructan were markedly altered in the mycorrhizal plants. In roots of non-fertilized mycorrhizal plants, fructan pools were significantly greater than in the corresponding non-mycorrhizal plants. By contrast, fertilization caused a general decrease in amounts of fructan in roots. The increase of fructan in mycorrhizal roots was correlated with a decrease of invertase activity. In leaves, fructan pools decreased or remained unchanged upon mycorrhizal infection; fertilization had a similar effect. However, when individual leaves of a plant were compared, intriguing effects of the mycorrhizal symbiosis could be observed. Whereas in non-mycorrhizal plants, the youngest leaves had the highest fructan contents and the oldest leaves the lowest (as previously reported), this gradient was markedly altered in mycorrhizal plants, indicating systemic effects of mycorrhiza on assimilate partitioning in shoots.

The size and distribution of Cortinarius rotundisporus genets at three sclerophyll forest field sites in New South Wales, Australia, were estimated by using microsatellite-primed PCR (MS-PCR) of DNA extracted from sporocarp tissue. MS-PCR fingerprints generated with the primers (GTG)5 and (GACA)4 indicated that two to five genets were present at each site, with each site being characterized by a single large genet (9–30 m in diameter). Analysis of internal transcribed spacer (ITS)-RFLP patterns from individual sporocarps used in the study suggested that three distinct RFLP types were present in the sampled C. rotundisporus population. ITS sequence data indicate that the three RFLP types had less than 88.4% sequence identity to each other, strongly suggesting that C. rotundisporus is a complex of three species.

An experiment was performed to find out whether ectomycorrhizal (ECM) fungi alter the nitrogen (N) isotope composition, δ15N, of N during the transport of N from the soil through the fungus into the plant. Non- mycorrhizal seedlings of Pinus sylvestris were compared with seedlings inoculated with either of three ECM fungi, Paxillus involutus, Suillus bovinus and S. variegatus. Plants were raised in sand in pots supplied with a nutrient solution with N given as either NH4+ or NO3−. Fractionation against 15N was observed with both N sources; it decreased with increasing plant N uptake, and was larger when NH4+ was the source. At high ratios of Nuptake/Nsupplied there was no (NO3−), or little (NH4+), fractionation. There seemed to be no difference in fractionation between ECM and non-mycorrhizal plants, but fungal rhizomorphs were sometimes enriched in 15N (up to 5‰ at most) relative to plant material; they were also enriched relative to the N source. However, this enrichment of the fungal material was calculated to cause only a marginal decrease (−0.1‰ in P. involutus) in δ15N of the N passing from the substrate through the fungus to the host, which is explained by the small size of the fungal N pool relative to the total N of the plant, i.e. the high efficiency of transfer. We conclude that the relatively high 15N abundance observed in ECM fungal species should be a function of fungal physiology in the ECM symbiosis, rather than a reflection of the isotopic signature of the N source(s) used. This experiment also shows that the δ15N of plant N is a good approximation of δ15N of the available N source(s), provided that N is limiting growth.

The use of biofuels has been proposed as one possible substitute for fossil fuels, which contribute substantially to the increase in [CO2] in the atmosphere. However, increased harvesting of forest residues for biofuel might affect the availability of base cations, P and N, as well as the development, community dynamics and function of ectomycorrhizas. This in turn might influence nutrient uptake and tree growth. In this study we investigated the effects of repeated forest residue harvesting on ectomycorrhizal species colonizing spruce roots in the humus layer of a 35-yr-old forest. Harvesting significantly decreased the thickness of the humus layer as well as decreasing the numbers of ectomycorrhizal root tips both per metre root length and per unit humus volume. Changes in mycorrhizal community structure were studied by ITS typing with the use of PCR–RFLP analysis. In total, 19 different ITS types were found on two different sampling occasions (autumn and spring); 11 of these were common to both samplings. Nine of the ITS types were identified to at least the genus level by comparison with RFLP patterns of identified fruiting bodies or axenic cultures. Five species, Cortinarius sp. 2, Thelephora terrestris (Ehrenb.) Fr., Lactarius theiogalus (Bull.[ratio ]Fr.) S. F. Gray s.st. Neuhoff, Tylospora fibrillosa Donk and Tö-96-12, occurred on over 5% of the total sampled root tips. Together these five types colonized 63% of the mycorrhizas screened. A similarity index assessment showed no shift in mycorrhizal community structure as a result of harvesting. Our findings suggest that the repeated removal of forest residues might have a strong effect on the quantity and development of ectomycorrhizal roots in the organic horizon, but little effect on the species composition of the community.