It is easy to dismiss bryophytes as ‘lower’ plants, mere primitive precursors long since left behind in the evolutionary race, and of only rather esoteric and incidental biological interest. But this is to let oneself be led astray by a simplistic image of a tidy evolutionary tree – an image that served Darwin well a century and a half ago (Desmond & Moore, 1992), but which we should now see as an intricately branched evolutionary bush with innumerable shoots reaching out from all depths to the growing apices that represent the present day. The earliest land plants may indeed have been at a bryophyte level of organization, but modern bryophytes, no less than vascular plants, are the product of some 450 million years’ evolution since that time (Edwards et al., 1998). Raven (1977, 1984) has emphasized the importance of the evolution of supracellular transport systems in the origin of vascular land plants. Bryophytes, on the other hand, evolved desiccation tolerance and represent an alternative strategy of adaptation to life on land, photosynthesizing and growing when water is available, and suspending metabolism when it is not. They are limited by their mode of life, but also liberated : they are prominent on hard substrates such as rock and bark, which are impenetrable to roots and untenable to vascular plants. Bryophytes (in species numbers the second biggest group of green land plants) may be seen as the mobile phones, notebook computers and diverse other rechargeable battery-powered devices of the plant world – not direct competitors for their mains-based equivalents, but a lively and sophisticated complement to them.

‘The view that nutrient acquisition by most plants growing in natural ecosystems is mediated by mycorrhiza-forming symbiotic fungi is now largely accepted’ (Read, 2000). Is this bold claim really true for the whole suite of mineral nutrients that plants require? The case is strongest for nutrients that are not very mobile in soil, especially when present in growth-limiting amounts, and phosphate (P) is the classic example. Arbuscular mycorrhizas are by far the most widespread mycorrhizal symbioses, and the ability of arbuscular mycorrhizal (AM) fungi to take up soil nutrients such as P and transfer them to the host plant is an area of intense research. However, there is great variation in the extent to which AM plants benefit in measurable terms from the symbiosis under a given set of environmental conditions, and a paper in this issue, by Koide et al., addresses this problem (Koide et al., pp. 163–168). The variability is especially apparent in the field, thus obscuring the possible roles of mycorrhizas in community structure and succession (Fitter, 1985; McGonigle, 1988).

John Innes Centre, Norwich, UK, June 2000

Genomics research involving legumes, an area that is attracting major funding, has two distinct branches – work involving model species, and work involving crops. This meeting aimed to stimulate communication between these two groups. The major research areas covered included leaf, flower and seed development, establishment of symbioses, pathogen interactions and applied aspects (from the conservation of legume ecotypes to products required by the market).

Summary 11

I. INTRODUCTION 12

II. LICHEN BIONT CHARACTERISTICS 12

1. The mycobiont 12

2. The photobiont 13

III. LICHEN GROWTH 13

1. Allocation of resources 13

2. Growth rates and environmental limitations 14

3. Maintaining an optimal energy use efficiency 15

IV. CARBON ACQUISITION 16

1. Water relations 16

(a) Desiccation tolerance 16

(b) Activation upon re-hydration 17

(c) Diffusion of water and CO2 18

2. Photobiont CO2fixation 20

(a) CO2 acquisition modes 20

(b) Significance of the CCM 22

3. Light and nitrogen relations 22

(a) The light-response curve 23

(b) Photosynthetic capacity 25

(c) Coping with high light 27

V. CARBON SINKS AND EXPENDITURES 28

1. Carbon translocation 28

2. Carbon sinks 29

3. Respiration 30

VI. CONCLUDING REMARKS 30

Acknowledgements 31

References 31

Lichens are nutritionally specialized fungi (the mycobiont component) that derive carbon and in some cases nitrogen from algal or cyanobacterial photobionts. The mycobiont and photobiont live together in an integrated thallus, but they lack specific tissue for the transport of metabolites and resources between them. Carbon is acquired through photosynthesis in the photobiont, which is active when the lichen is wet and exposed to light. Lichen photosynthesis is limited primarily by water, light and nitrogen, but can also be constrained by slow diffusion of CO2 within the wet thallus. The assimilated carbon is exported from photobiont to mycobiont, which also predominates in terms of biomass, and apparently regulates the size of the photobiont population. It has therefore generally been assumed that most of the carbon is used for growth and maintenance of the fungal hyphae. However, the extent of photobiont respiration in relation to mycobiont respiration has seldom been quantified; neither do we know the pool sizes of various carbon sinks within lichens. Owing to this lack of fundamental data we do not know whether, or how, carbohydrate resources are regulated to maintain an optimal balance between energy input and expenditures in these symbiotic organisms. This review summarizes data on growth, carbon gain and carbon expenditures in lichens, with particular emphasis on factors determining the photosynthetic capacity of their photobionts. An attempt is made to introduce an economic analysis of lichen growth processes, a view that has often been adopted in studies of higher plants. Areas in which more data are needed for the construction of a model on ‘lichen resource allocation patterns’ are discussed.

Summary 37

I. INTRODUCTION 37

II. DIGESTION OF PLANTS IN THE RUMEN: OLD AND NEW CONCEPTS 39

III. RUMEN-INDUCED PLANT METABOLISM: CELL DEGRADATION AND DEATH 41

1. Summary of plant cell death processes 41

2. Anaerobic stress and flooding tolerance of plants 42

3. Plant cell responses to elevated temperatures 45

4. Wounding responses/pathogen attachment 45

5. Senescence in the rumen? 47

IV. FUTURE PROSPECTS 50

Acknowledgements 51

References 51

It is generally assumed that breakdown of plant material in the rumen is a process mediated by gut microorganisms. This view arose because of the identification of a pre-gastric fermentation in the rumen, brought about by a large and diverse microbial population. The extensive use of dried and ground feed particles in forage evaluation might have helped to promote this assumption. However, although the assumption might be correct in animals feeding on conserved forage (hay and silage) where the cells of ingested forage are dead, it is possible that with grazed (living) forage, the role played by plant enzymes in the rumen has been overlooked. In a grazing situation, plant cells that remain intact on entering the rumen are not inert, but will respond to the perceived stresses of the rumen environment for as long as they are metabolically viable. Metabolic adjustments could include anaerobic and heat-shock responses that could promote premature senescence, leading to remobilization of cell components, especially proteins. Moreover, contact of plant cells with colonizing microorganisms in the rumen might promote a type of hypersensitive response, in much the same way as it does outside the rumen. After fresh plant material enters the rumen and prior to extensive plant cell-wall degradation, there is often a phase of rapid proteolysis providing N in excess of that required to maintain the rumen microbial population. The inefficient use of this ingested N results in generation of ammonia and urea in exhaled breath and urine, which promotes welfare and environmental pollution concerns. Therefore an important research goal in livestock agriculture is to find ways of decreasing this initial rate of proteolysis in the rumen. This will benefit the farmer financially (through decreased use of feed supplements), but will also benefit the environment, as N pollution can adversely affect pasture diversity and ecology. This review considers the possible responses of plant metabolism to the rumen environment, and how such considerations could alter current thinking in ruminant agriculture.

The present study of structural and physiological changes during the development of the cushion moss, Grimmia pulvinata, quantifies the size-dependence of various parameters of water relations such as changes in surface: volume ratio (S/V) or water loss rates, and also measures net CO2 gas exchange in the light and the dark. Larger cushions had lower S/V values than smaller ones and featured lower rates of area-based evapotranspiration, owing to higher boundary-layer resistance, but did not differ in relative water storage capacity (expressed as a percentage of d. wt). In combination, this leads to considerably longer hydration periods in larger cushions. By contrast, CO2 gas-exchange parameters were negatively correlated with size : larger cushions showed significantly lower (mass-based) rates of net photosynthesis and dark respiration. Using these data, we estimated carbon budgets during a drying cycle as a function of cushion size. When including alternations of dark and light periods, the relationship proved to be rather complicated. Depending on the time of hydration, net carbon budgets not only varied quantitatively with size but sometimes took on both positive and negative values depending on cushion size. We conclude that neglecting plant size can lead to unrepeatable or even misleading results in comparative ecophysiological studies, and therefore urge for adequate attention to be paid to size in these studies.

Experiments were conducted on Phillyrea latifolia plants grown under a dense overstorey of Pinus pinea (shade plants) or on seashore dunes (sun plants) in a coastal area of Tuscany (42° 46′ N, 10° 53′ E). Total integrated photon flux densities averaged 1.67 and 61.4 m mol m−2 d−1 for shade and sun sites, respectively. A leaf morphological–structural analysis, a qualitative and quantitative analysis of phenylpropanoids of leaf tissue and leaf surface, and a histochemical localization of flavonoids were conducted. The area of sun leaves reached 57% of that of shade leaves, whereas leaf angle (β), sclerophylly index (ratio of leaf d. wt:leaf area), and trichome frequency (trichome number mm−2 ) were markedly greater in leaves exposed to full solar radiation than in leaves acclimated to shade. The total thickness of sun leaves was 78% higher than that of shade leaves, mostly owing to a greater development of both palisade parenchyma and spongy mesophyll. The concentration, but not the composition, of leaf tissue phenylpropanoids varied significantly between sun and shade leaves, with a marked increase in flavonoid glycosides in sun leaves. Flavonoids occurred almost exclusively in the upper epidermal cells of shade leaves. By contrast, flavonoids largely accumulated in the upper and lower epidermis, as well as in the mesophyll tissue of leaves that were acclimated to full sunlight. Flavonoid glycosides were found exclusively in the secretory products of glandular trichomes of P. latifolia leaves exposed to high levels of light; luteolin 7-O- glucoside and quercetin 3-O-rutinoside were the major constituents. By contrast, verbascoside and an unidentified caffeic acid derivative constituted 72% of total phenylpropanoids secreted by glandular trichomes of shade leaves, whereas they were not detected in glandular trichomes of sun leaves. These findings suggest that the light-induced synthesis of flavonoids in glandular trichomes of P. latifolia probably occurs in situ and concomitantly inactivates other branch pathways of the general phenylpropanoid metabolism. This is the first report of the key role of glandular trichomes and of flavonoid glycosides in the integrated mechanisms of acclimation of P. latifolia to excess light.

We have explored leaf-level plastic response to light and nutrients of Quercus ilex and Q. coccifera, two closely related Mediterranean evergreen sclerophylls, in a factorial experiment with seedlings. Leaf phenotypic plasticity, assessed by a relative index (PI = (maximum value - minimum)/maximum) in combination with the significance of the difference among means, was studied in 37 morphological and physiological variables. Light had significant effects on most variables relating to photosynthetic pigments, chlorophyll fluorescence and gas exchange, whereas nutrient treatment had a significant effect in only 10% of the variables. Chlorophyll content was higher in the shade whereas carotenoid content and nonphotochemical quenching increased with light. Nutrient limitations increased the xanthophyll-cycle pool but only at high light intensities, and the same interaction between light and nutrients was observed for lutein. Predawn photochemical efficiency of PSII was not affected by either light or nutrients, although midday photochemical efficiency of PSII was lower at high light intensities. Photosynthetic light compensation point and dark respiration on an area basis decreased with light, but photosynthetic capacity on a dry mass basis and photochemical quenching were higher in low light, which translated into a higher nitrogen use efficiency in the shade. We expected Q. ilex, the species of the widest ecological distribution, to be more plastic than Q. coccifera, but differences were minor: Q. ilex exhibited a significant response to light in 13% more of the variables than Q. coccifera, but mean PI was very similar in the two species. Both species tolerated full sunlight and moderate shade, but exhibited a reduced capacity to enhance photosynthetic utilization of high irradiance. When compared with evergreen shrubs from the tropical rainforest, leaf responsiveness of the two evergreen oaks was low. We suggest that the low leaf-level responsiveness found here is part of a conservative resource use strategy, which seems to be adaptive for evergreen woody plants in Mediterranean-type ecosystems.

The flooding tolerance of Carex species was studied in relation to their field distribution and their capacity to form root aerenchyma under controlled conditions. In an alpine meadow, six Carex species were selected which were distributed in a clear zonation correlating with water content of the soil. Carex sempervirens and C. ferruginea were only found on nonflooded soil, the latter species preferring moister conditions. Carex davalliana and C. nigra were both associated with water-saturated soil, whereas C. limosa and C. rostrata preferred partially submerged conditions. Carex davalliana and C. limosa were bound to flooded soils with a relatively high redox potential and horizontally flowing groundwater. Carex rostrata and C. nigra grew in stagnant soil-flooded conditions with low soil redox potentials. The amount of aerenchyma in the roots of all species increased when grown in oxygen-deficient stagnant agar. This increase in root porosity, combined with increased root diameter, presumably improved internal aeration of the roots. Although all species survived experimental soil flooding, partial submergence was lethal to C. sempervirens and, surprisingly, also to the wetland species C. davalliana. Carex ferruginea showed a reduced growth rate during partial submergence. The three other species, all wetland plants, reached highest biomass production under soil-flooded and partially submerged conditions, with slower growth on free-draining soil. It is concluded that aerenchyma is not constitutive in the Carex species under study, and is best developed in Carex species from wetlands. Species with less aerenchyma perform poorly when soil-flooded, but conditions of partial submergence could even affect species with a considerable amount of root aerenchyma.

Spore capsules of four Sphagnum species were buried at different depths in peat on a bog. Spore viability was determined after 0, 1, 2 and 3 yr. Viability generally declined with time, but viable spores were still found at all depths after 3 yr. The light-coloured spores of S. balticum and S. tenellum retained their viability better than the darker spores of S. fuscum and S. lindbergii. Survival was highest under wet but aerobic conditions, but was also high under humid or periodically desiccated conditions. By contrast, most spores stored under wet, anaerobic conditions died within 2–3 yr. These results, and predictions from them, are not consistent with earlier results for spores of long-lived and dominant bryophytes, or for seeds of phanerogams of undisturbed wetlands and forests. There was no correlation between spore size and longevity across species, but the small spores from small capsules of S. balticum and S. tenellum generally showed higher viability than those from the medium-sized and large capsules of the same species. This suggests a positive intraspecific relationship between longevity and dispersal distance. There was an indication of conditional dormancy, controlled by weather, in Sphagnum spores. The experiments indicate that Sphagnum spores can form a long-term persistent spore bank under suitable conditions, with a half-life of between 1 and 20 yr (mean across species of 2.6 and 5.0 yr at two depths studied), and with potential values in individual spore capsules of several decades, or even of centuries. Sphagnum spores kept refrigerated showed 15–35% viable spores after 13 yr. The capacity to form a persistent spore bank that can be activated whenever favourable conditions occur might help explain the wide geographical distribution of many Sphagnum species in the boreal and temperate zones, where they have managed to colonize almost every suitable patch of acidic, nutrient-poor wetland.

The first known indicator of probable fern epiphytism in the Tertiary fossil record is documented from the Eocene London Clay of southeast England. This pyritised fern rachis exhibits major deviation from the basic form of petiolar vascular trace morphology shown by the other London Clay fossil ferns and is here attributed to the Polypodiaceae. This represents the first known occurrence of this family in the London Clay macroflora, and brings the total of distinct fern rachis types from the Lower Tertiary of southeast England to six. The significance of this find is interpreted in relation to its probable origins from a tropical rainforest palaeoflora in which it is proposed that this specimen represents the first occurrence of an epiphytic component.

Using the outcrossing Primula farinosa and its autogamous selfing relatives P. scotica, P. scandinavica and P. stricta, we compared the fitness of light and heavy seeds. Heavy seeds germinated in greater numbers and more quickly. In competition with seedlings grown from lighter seeds, heavy seeds produced larger rosettes. In P. farinosa such seedlings went on to produce more seeds, and in two populations heavier seeds, than plants from lighter seeds. After transplantation to natural populations, seedlings of P. farinosa derived from heavy seeds produced larger rosettes, more flowers and seeds than those from lighter seeds in certain populations so that seedlings born of heavy seeds were much fitter than seedlings from lighter seeds. Average seed weight varied in inverse proportion to seed number per capsule. The autogamous species produced on average about twice as many seeds per capsule as P. farinosa. In P. scotica and P. stricta this difference appears to be due in part to assured fertilization, but this high fecundity did not cause disadvantageously light seeds. As these species produced fewer capsules per scape, their overall seed production was on average no greater than for P. farinosa. P. farinosa traded-off fitness between capsules with large seed numbers, which donated more offspring to the next generation, and those with small seed numbers, whose heavy seeds would be more likely to reproduce themselves in the next generation. We conclude that low fecundity in outcrossing species might at times be advantageous.

Experiments were set up to assess the relative impacts of grass lamina fragment density, size and quality on the activity and growth of four fungi in a particulate soil-like matrix. The fungi studied were Cladosporium cladosporioides, Fusarium lateritium, Phoma exigua and Trichoderma viride, all of which are common inhabitants of UK upland grassland soils. Resource quality was varied by using three contrasting grasses as sources of lamina fragments: Lolium perenne, Agrostis capillaris and Nardus stricta. All the fungi were able to forage effectively through the soil-like matrix (at a rate of 1–2 ml matrix d−1) and colonize and partially decompose available lamina fragments (up to 40% d. wt loss). Foraging rates (ml matrix d−1) were affected by lamina fragment species but not by fragment density or size. In general, F. lateritium and T. viride foraged at a faster rate than the other species. Mycelial activity in the soil-like matrix was directly proportional to total lamina fragment availability and was unaffected by fragment size. Biomass production on solid media was also directly related to substrate (carbohydrate) availability. The results indicate that these fungi can adjust their growth patterns in response to fragment density so as to maximize foraging efficiency (energy acquired per unit exploratory biomass production). Differences in lamina fragment species were responsible for up to 50% reduction in mycelial activity.

A total of 107 putative ericoid mycorrhizal endophytes were isolated from hair roots of Calluna vulgaris from two abandoned arsenic/copper mine sites and a natural heathland site in southwest England. The endophytes were initially grouped as 14 RFLP types, based on the results of ITS-RFLP analysis using the restriction endonucleases Hinf I, Rsa I and Hae III. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with sequences for known ericoid mycorrhizal endophytes and selected ascomycetes. The majority of endophyte isolates (62–92%) from each site were identified as Hymenoscyphus ericae, but a number of other less common mycorrhizal RFLP types were also identified, all of which appear to have strong affinities with the order Leotiales. None of the less common RFLP types was isolated from C. vulgaris at more than one field site. Neighbour-joining analysis indicated similarities between the endophytes from C. vulgaris and mycorrhizal endophytes isolated from other Ericaceae and Epacridaceae hosts in North America and Australia.

Two mycotrophic species (Lactuca sativa and Abutilon theophrasti) and one nonmycotrophic species (Beta vulgaris) were grown in a P-deficient soil, and the effects of mycorrhizal inoculation on three variables that determine growth rate were assessed for each. The phosphorus-use efficiency (PUE, dW/dP) is the ratio of d. wt increase to P content increase. Plant P is the amount of P (the limiting resource) controlled by the plant, which can be allocated to various purposes. The phosphorus efficiency index (PEI, dP/Pdt) is the efficiency with which plant P is used to acquire P from the soil. Inoculated and control plants of a given species initially contained the same amount of P because all plants were grown from seed. Mycorrhizal colonization significantly increased the PEI of Lactuca and Abutilon (by 23 and 32%, respectively). As expected, mycorrhizal inoculation did not significantly increase the PEI of Beta. As a result, mycorrhizal inoculation significantly increased the P content of Lactuca and Abutilon, but not Beta. Mycorrhizal colonization decreased the PUE of lettuce, but did not significantly affect that of Abutilon or Beta. Mycorrhizal inoculation therefore slightly increased the growth rate of Lactuca, greatly increased the growth rate of Abutilon, and ultimately had no significant effect on the growth rate of Beta.

A study was conducted to quantify the ability of entrapped, monoxenically produced spores of an arbuscular mycorrhizal fungus to germinate and reproduce the fungal life cycle after cryopreservation. No germination was obtained after incubation of entrapped spores in glycerol and mannitol and subsequent cryopreservation at −70 °C, regardless of the concentration of cryoprotectants and duration of incubation. Incubation for 1 d in 0.5 M sucrose, and for 1 and 2 d in 0.5 M trehalose, led to spore germination after cryopreservation at −70 °C. Lower cryopreservation temperatures were tested with entrapped spores incubated for 1 d in 0.5 M trehalose. The highest germination rate, estimated by the percentage of potentially infective beads (%PIB), was obtained at −100 °C. A %PIB of 95% (water agar medium) to 100% (Strullu–Romand medium) was obtained at this temperature. Thereafter, %PIB rapidly decreased at −140 and −180 °C. Heavy sporulation and high internal root colonization were obtained after re-association of the entrapped spores, incubated for 1 d in 0.5 M trehalose and subsequently cryopreserved at −100 °C, with transformed carrot roots. This demonstrates the ability of entrapped spores to reproduce the fungal life cycle following cold treatment.

The effect of colonization of tissue-cultured strawberry (Fragaria × ananassa Duch. cv. Kent) plantlets in vitro by the arbuscular mycorrhizal fungus (AMF) Glomus intraradices on plantlet response to poly(ethylene glycol) (PEG)-8000-induced water stress was investigated. The plantlets were inoculated axenically and co-cultured with the AMF for 4 wk, then transferred to 15% PEG-8000 solutions for 4, 8 and 12 h. Relative water content, water potential, osmotic potential, leaf conductance for water vapour diffusion and photosynthetic efficiency as estimated by chlorophyll a fluorescence were all affected by the PEG treatment and its duration but not by the presence of the intraradical phase of the AMF. However, distinct differences in PEG-induced changes in amino acid content were observed between nonmycorrhizal and mycorrhizal plantlets. In the latter, the treatment with PEG caused a substantial decrease in asparagine levels in leaves that was accompanied by a marked increase in asparagine concentration in roots. The opposite was observed in nonmycorrhizal plantlets. Furthermore, concentrations of aspartic acid, serine, threonine, amino-N-butyric acid, alanine and starch increased in roots of mycorrhizal and decreased in nonmycorrhizal plantlets. Our results suggest the presence of a mobile pool of asparagine that can be translocated from leaves to roots or vice versa in response to PEG-induced water stress, depending on the mycorrhizal status of the plantlets. These opposite patterns suggest different strategies of mycorrhizal and nonmycorrhizal plantlets to water stress, which seem to involve different adjustments in nitrogen and carbon metabolism.