The number of papers submitted to New Phytologist by authors working in North America has increased significantly in recent years. We are now able to offer those authors a better service because, from this month, they will be able to submit their papers direct to our new USA Office – this is being supervised by one of our Editors, Richard Norby, and is located at Oak Ridge, Tennessee:

New Phytologist USA Office, Oak Ridge National Laboratory, Bldg 1059, PO Box 2008, Oak Ridge, TN 37831-6422, USA. Tel: +1 423 576 5261. Fax: +1 423 576 9939. E-mail: [email protected].

Summary 3

I. introduction 4

II. the cyanobacteria 7

III. the heterocyst 9

1. Function and metabolism 9

2. Heterocyst structure 12

(a) Overview 12

(b) The polysaccharide (homogeneous) layer 12

(c) The glycolipid (laminated) layer 12

(d) The septum and microplasmodesmata 12

3. Nitrogen regulation and heterocyst development 12

4. Heterocyst development 13

(a) The proheterocyst 13

(b) Proteolysis associated with heterocyst development 14

(c) RNA polymerase sigma factors 14

(d) Developmental regulation of heterocyst cell wall and nitrogenase gene expression 14

(e) Genome rearrangements associated with heterocyst development 15

5. Genes essential for heterocyst development 15

(a) hetR 15

(b) Protein phosphorylation and the regulation of hetR activity 16

(c) hetR in nonheterocystous cyanobacteria 16

(d) Other heterocyst-specific genes 16

6. Heterocyst spacing 18

(a) Patterns of heterocyst differentiation 18

(b) Genes involved in heterocyst spacing 18

(c) Disruption of heterocyst pattern 18

7. Filament fragmentation and the regression of developing heterocysts 20

8. The nature of the heterocyst inhibitor 20

9. Cell selection during differentiation and pattern formation 20

(a) Cell division 20

(b) DNA replication and the cell cycle 21

(c) Competition 21

10. Models for heterocyst differentiation and pattern control 21

IV. the akinete 23

1. Properties of akinetes 23

2. Structure, composition and metabolism 24

3. Relationship to heterocysts 24

4. Factors that influence akinete differentiation 24

5. Extracellular signals 25

6. Akinete germination 25

7. Genes involved in akinete differentiation 26

V. conclusion 26

Acknowledgements 27

References 28

Cyanobacteria are an ancient and morphologically diverse group of photosynthetic prokaryotes. They were the first organisms to evolve oxygenic photosynthesis, and so changed the Earth's atmosphere from anoxic to oxic. As a consequence, many nitrogen-fixing bacteria became confined to suitable anoxic environmental niches, because the enzyme nitrogenase is highly sensitive to oxygen. However, in the cyanobacteria a number of strategies evolved that protected nitrogenase from oxygen, including a temporal separation of oxygenic photosynthesis and nitrogen fixation and, in some filamentous strains, the differentiation of a specialized cell, the heterocyst, which provided a suitable microaerobic environment for the functioning of nitrogenase. The evolution of a spore-like cell, the akinete, almost certainly preceded that of the heterocyst and, indeed, the akinete may have been the ancestor of the heterocyst. Cyanobacteria have the capacity to differentiate several additional cell and filament types, but this review will concentrate on the heterocyst and the akinete, emphasizing the differentiation and spacing of these specialized cells.

The relationships of foliage assimilation capacity per unit area (PPmax) with leaf dry mass per unit area (LMA) and nitrogen content per unit area (NP) differ between species and within species grown in different habitats. To gain a more mechanistic insight into the dependencies of PPmax on LMA and NP, this literature study based on 597 species from a wide range of earth biomes with woody vegetation examines the relations between leaf photosynthetic capacity and the components of LMA (leaf density (D, dry mass per volume) and thickness (T)), and also the correlations of D and T with leaf nitrogen content and fractional leaf volumes in different tissues. Across all species, PPmax varied 12-fold and photosynthetic capacity per unit dry mass (Pmmax) 16-fold, NP 12-fold, and nitrogen per unit dry mass (Nm) 13-fold, LMA 46-fold, D 13-fold, and T 35-fold, indicating that foliar morphology was more plastic than foliar chemistry and assimilation rates. Although there were strong positive correlations between PPmax and NP, and between Pmmax and Nm, leaf structure was a more important determinant of leaf assimilation capacities. PPmax increased with increasing LMA and T, but was independent of D. By contrast, Pmmax scaled negatively with LMA because of a negative correlation between Pmmax and D, and was poorly related to T. Analysis of leaf nitrogen and tissue composition data indicated that the negative relationship between D and Pmmax resulted from negative correlations between D and Nm, D and volumetric fraction of leaf internal air space, and D and symplasmic leaf fraction. Thus, increases in leaf density bring about (1) decreases in assimilative leaf compounds, and (2) extensive modifications in leaf anatomy that may result in increases in intercellular transfer resistance to CO2. Collectively, (1) and (2) lead to decreased Pmmax, and also modify PPmax versus LMA relationships.

Clones of ectomycorrhizal fungi can colonize new areas through production of vegetative mycelium or spore dispersal, but the relative importance of these processes in nature is not known. In this study, sporocarps of an ectomycorrhizal fungus, Suillus grevillei, were mapped and sampled from a Larix kaempferi stand at the foot of Mt Fuji. DNA was extracted directly from each sporocarp, and DNA polymorphism was analysed by polymerase chain reaction (PCR) amplification of inter-simple sequence repeat (ISSR) regions primed by (GTG)5, (GCC)5 and (GACA)4. Different sensitivities to detect polymorphism were found among the three primers, with (GACA)4 showing the highest sensitivity. Forty seven sporocarps were analysed by the three ISSR primers and divided into 34 genets based on combination of PCR fingerprints. In the population 28 genets were represented by individual sporocarps. In most cases, sporocarps grown in aggregation (within a circle of 50 cm diameter) showed some different ISSR band patterns. These results suggest that genets of S. grevillei at the test site are relatively small. The genetic similarities between the 34 genets were also calculated and similarity groups were determined by the criterion that all similarity F values of genets within a group were not <80%. In general, the genets within a similarity group located close to each other. The results of multiple different but highly related genets in a small area suggest that the population of S. grevillei in this stand is not spread and maintained by clonal mycelium extension but is reproduced by spore dispersal.

Sunflower seedlings (Helianthus annuus hybrid Select) were grown in a complete nutrient solution in the absence or presence of Cd2+ (10 and 20 μM). Analyses were performed to establish whether there was a differential effect of Cd2+ on mature and young leaves. After 7 d the growth parameters as well as the leaf area had decreased in both mature and young leaves. Accumulation of Cd2+ in the roots exceeded that in the shoots. Seedlings treated with Cd2+ exhibited reduced contents of chlorophyll and CO2 assimilation rate, with a greater decrease in young leaves. The photochemical efficiency of photosystem II (PSII) was not altered by Cd2+ treatment in either mature or young leaves, although during steady-state photosynthesis in young leaves there was a significant alteration in the following parameters: quantum yield of electron transport by PSII (ΦPSII), photochemical quenching (qP), non-photochemical quenching (qNP), and excitation capture efficiency of PSII (Φexc).

The effects of excess nitrogen (N) on the frost hardiness and freezing injury of bark and buds were tested in 2-yr-old sessile oaks (Quercus petraea) and pedunculate oaks (Q. robur) that had been grown outdoors in sand culture with normal or luxurious N supply during the growing season. Some trees from both N treatments were subjected to drought stress in summer, whereas others were adequately watered. Between January and March, whole plants were exposed to artificial freezing treatments at −25 °C of different durations (10 d, 21 d, or two periods of 4 d interrupted by an 8 d frost-free period, referred to as a freeze–thaw cycle). The frost hardiness and freezing injury of the bark were determined by two different versions of the electrolyte leakage method (calculation of an index of injury, I−25, from the relative conductivity after a fixed time; and measurement of the electrolyte leakage rate). In addition, the vitality of bark and buds was assessed visually with the 2,3,5-triphenyltetrazolium chloride (TTC) method. In comparison with the control, the oaks that had been luxuriously supplied with N exhibited distinctly higher N concentrations and lower C:N ratios in their leaves and bark, higher relative shoot increments, delayed leaf discoloration in autumn, and earlier budbreak in spring. Oaks kept outdoors during the winter were frost-hardy from December until the end of February without showing differences between species or N treatments. In comparison with the pedunculate oak, the sessile oak was more susceptible to freezing injury induced by prolonged artificial freezing or by the freeze–thaw cycle. Increased freezing injury in trees with high N supply was detected only by the TTC test of the bark of sessile oaks subjected to the freeze–thaw cycle. In all oaks, except for the pedunculate oaks grown with normal N, the freeze–thaw cycle resulted in significantly lower frost hardiness, as indicated by increased I−25 values. This treatment also led to increased freezing damage of bark and buds, as revealed by the TTC test. In drought-stressed oaks, the electrolyte leakage rate was significantly elevated after the freeze–thaw cycle. It is concluded that, in the sessile and pedunculate oaks, the water supply before frost stress and the course of the temperature in winter have a greater effect on frost hardiness and freezing damage than excess N.

Most recent reviews of plant salinity response have included the concept of a nutritional disturbance as one likely mechanism by which shoot growth might be inhibited. None the less, few studies of dicotyledonous plants have presented data on nutrient transport into the most intensively growing shoot tissues. In this paper net nutrient deposition was followed for 3 d in 8 sequential, growing leaves of Lactuca sativa, which were grown either in conditions of moderate salinization, or in a growth-stimulating concentration of NaCl. The nutrient deposition was studied from 0.7 to 3.7 d following completion of stepwise salinization. This deposition was followed in immature leaves, which had attained only 1–2% of ultimate leaf mass by the completion of the study. In such young leaves development is still dominated by cell division. The transport of Ca2+ specifically to the youngest leaves was reduced by more than twice as much as was K+ transport. Transport of the other major divalent cationic nutrient, Mg2+, was not decreased for these leaves. The factors of increase for Na+ and Cl− after 3.7 d after completion of salinization averaged 152 and 62% over control levels for the three youngest leaves (for Na+ and Cl−, respectively). Though significant, these increases were only 27 and 14% as great as increases in three leaf sets of more developed growing leaves. Decreases in net K+ deposition and leaf K+ concentration were not greater for the youngest than they were for the oldest leaves. Net S deposition was reduced 44% more in younger than older growing leaves, but for most leaves not beyond the level expected due to reduced sink strength. The reduction in net P deposition also seemed more related to reduced sink strength, but was reduced to approx. 50% in both younger and more developed growing leaves. While Fe concentration was not reduced by salinization at any developmental stage, Zn2+ net transport and Zn concentration were both reduced in the two youngest leaves (57 and 70%, respectively). Given the moderate treatment imposed (Na:Ca ratio of 22) the results suggest that Ca2+ transport to the youngest leaves is probably highly sensitive to salinization of the root medium and is perhaps a key physiological response in the inhibition of leaf growth.

Young trees of European beech (Fagus sylvatica) acclimated for one growing season to ambient (c. 367 μl l−1) or elevated CO2 levels (c. 660 μl l−1) were exposed during the subsequent year to combinations of the same CO2 regimes and ambient or twice-ambient ozone (O3) levels (generated from the database of a rural site). By the end of June, before the development of macroscopic leaf injury, the raised O3 levels had not affected the light and dark reactions of photosynthesis. However, acclimation to elevated CO2 had resulted in lowered chlorophyll and nitrogen concentrations, whereas photosynthetic performance, examined over a wide range of parameters from light and dark reactions, remained unchanged or showed only slight reductions (e.g. apparent electron transport rate, ETR; apparent quantum yield of CO2 gas exchange, ΦCO2; apparent carboxylation efficiency, CE; and photosynthetic capacity at light and CO2 saturation, PC). In August, after the appearance of leaf necroses, plants grown under ambient CO2 and twice-ambient O3 conditions declined in both the photosynthetic light reactions (optimum electron quantum yield, Fv/Fm, non-photochemical energy quenching, NPQ, reduction state of QA, apparent electron quantum yield, ΦPSII, maximum electron transport rates) and the dark reactions as reflected by CE, ΦCO2, as well as the maximum CO2 uptake rate (i.e. PC). CE, ΦCO2 and PC were reduced by c. 75, 40 and 75%, respectively, relative to plants exposed to ambient CO2 and O3 levels. By contrast, plants exposed to twice-ambient O3 and elevated CO2 levels maintained a photosynthetic performance similar to individuals grown either under ambient CO2 and ambient O3, or elevated CO2 and ambient O3 conditions. The long-term exposure to elevated CO2 therefore tended to counteract adverse chronic effects of enhanced O3 levels on photosynthesis. Possible reasons for this compensatory effect in F. sylvatica are discussed.

The effects of summer drought, dew deposition on leaves and autumn rainfall on plant water relations and diurnal variations of photosynthesis were measured in two evergreen shrubs, rosemary (Rosmarinus officinalis) and lavender (Lavandula stoechas), grown in Mediterranean field conditions. Withholding water for 40 d caused a similar decrease in predawn shoot water potential (ψpd) from c. −0.4 to c. −1.3 MPa in both species, but a 50% decrease in the relative leaf water content in L. stoechas compared with 22% in R. officinalis. A similar decrease in CO2 assimilation rates by c. 75% was observed in water-stressed plants of both species, although L. stoechas showed smaller photosynthesis: stomatal conductance ratio than R. officinalis (35 vs 45 μmol CO2:mol H2O). The relative quantum efficiency of photosystem II photochemistry also decreased by c. 45% at midday in water- stressed plants of both species. Nevertheless, neither L. stoechas nor R. officinalis suffered drought-induced damage to photosystem II, as indicated by the maintenance of the ratio Fv:Fm throughout the experiment, associated with an increase in the carotenoid content per unit of chlorophyll by c. 62% and c. 30%, respectively, in water-stressed plants. Only L. stoechas absorbed dew by leaves. In this species the occurrence of 6 d of dew over a 15-d period improved relative leaf water content by c. 72% and shoot water potential by c. 0.5 MPa throughout the day in water-stressed plants, although the photosynthetic capacity was not recovered until the occurrence of autumn rainfall. The ability of leaves to absorb dew allowed L. stoechas to restore plant water status, which is especially relevant in plants exposed to prolonged drought.

The effects of partial defoliation on photosynthesis, whole-seedling carbon allocation, partitioning and growth were studied for two species with contrasting foliar traits. Field-grown seedlings of deciduous Japanese larch (Larix leptolepis) and evergreen red pine (Pinus resinosa) were defoliated by hand in early summer for 2 consecutive years. In the first year (1990), seedlings were defoliated by removing the distal 0, 25, 50 or 75% of each needle. In the second year (1991), seedlings were defoliated either 0 or 50%, regardless of previous defoliation treatments. Defoliation had little effect on photosynthesis and starch concentration in whole seedlings of either species in the first year. In the second year, photosynthesis increased in both species in response to the 1991 defoliation treatment, and in red pine also increased in response to the 1990 defoliation treatment. Further, in 1991 both larch and pine had decreased whole-seedling total non-structural carbohydrate concentrations in all seedlings that were defoliated at least once over the 2-yr period. This decrease was noted mostly in the starch component of the non-structural carbohydrates, and was similar in both species. In 1991, biomass was similarly decreased in both species in response to 1991 defoliation. Both species showed overcompensation in total and component biomass in seedlings defoliated by 25% in 1990. Overall, the results do not support the widely held belief that evergreen trees are substantially more affected than deciduous trees by defoliation.

Bulbous rush (Juncus bulbosus) initiates plant colonization in extremely acid lakes resulting from coal mining operations. Various analytical techniques (methylene blue/agar method, Ti3+-citrate solution) X-ray diffraction (XRD), scanning electron microscopy (SEM), and Energy-dispersive X-ray (EDX) were used to assess the mechanisms and strategies employed by J. bulbosus to overcome the extreme conditions. The plant releases oxygen into the rhizosphere in turn increasing the redox potential and inducing iron oxide plaque formation. XRD showed that the iron oxide of the plaque is mainly goethite that has been developed in the presence of CO2; SEM showed that there is a micro-space between the roots and sand grains which is inhabited by microorganisms. Furthermore, SEM-EDX studies on internal iron distribution demonstrate that iron toxicity is delayed by the physiological and biochemical structure of the plant. It is suggested that J. bulbosus uses a variety of mechanisms and strategies (morphological, physiological and biochemical adaptation) which are mainly complementary and which interact with each other to help J. bulbosus to manage its growth and survival in an extreme environment.

Concentrations of starch in roots of seeder species of Erica from the Cape Floristic Region, South Africa were found to be considerably less than in resprouters. Shoot starch was highly variable but mean values were similar in both seeder and resprouter species of Erica. Three distinct patterns of starch storage in roots were recognized. All seeder species fell within definitions of Categories 1 (narrow major and minor parenchymatous rays, one to two cells wide with no inter-ray storage) or 2 (thick major rays up to seven cells wide and thin minor rays with small amounts of inter-ray storage) whereas resprouter species were consistently within Categories 2 or 3 (broad major and minor rays, up to eight cells wide and conspicuous inter-ray starch storage). Results are discussed in light of similar studies of the related Epacridaceae. ‘Mixed’ species (i.e. with seeder or resprouter individuals present, often in distinct populations) were always classified as belonging to Category 2. Studies of populations of three ‘mixed’ species confirmed that seeder forms had consistently lower amounts of root starch than resprouters. Rays of xylem parenchyma were the main sites for starch storage in roots of both seeders and resprouters and greater proportions of cross-sectional area of roots were consistently devoted to such storage tissues in resprouter forms of the three ‘mixed’ species. Analyses of a number of seeder and resprouter Erica coccinea populations showed that differences in amounts of realised and potential root starch storage are best explained by the effect of regeneration behaviour rather than by among-population variability.

It is generally accepted that larger seeds give rise to seedlings with better performance. On the other hand, the size that a seed reaches is genetically determined by at least two different traits ; the genetic variability of the developing embryo and the genetic variability of the maternal plant. Thus, the relative contributions of these two traits affect seedling performance by influencing seed size. In this paper, I investigate the effect of seed size on seedling performance in the Scots pine (Pinus sylvestris). From eight maternal plants, 50 seeds were planted in each of two soil types (800 seeds in total), and seedling performance was monitored for 1 yr. Seed mass proved to be highly constant within maternal plants. Soil type influenced emergence and survival; however, the effect of soil type differed depending on maternal origin. Seed mass was positively correlated with seedling emergence, although this relationship was not found for seedling survival or date of emergence. The initial growth of the shoot was also positively correlated with seed mass. However, after one growing season, seed mass had no effect on seedling performance, which depended exclusively on maternal origin. Nevertheless, the mean mass of seeds produced by plants was positively correlated with mean values of growth parameters. Thus, first-year seedling performance seems to be a maternal trait indirectly associated with seed size.

Two arbuscular mycorrhizal fungi, Scutellospora calospora and Glomus sp. ‘City Beach’, were grown in soil conditions suitable for colonization of Allium porrum. Effects on plant growth and phosphate uptake were examined. Isolates of S. calospora (including the one used here) have been shown by others to vary in their stimulation of plant growth, and appear to be inefficient in the transfer of P from the fungus to the host. Our hypothesis was that such isolates of S. calospora may be more aggressive colonizers than other fungi, thus preventing growth increases or producing strong growth depressions in the host plant. In fact, inoculation with either fungus increased growth of plants in their respective soils both low in P (P0) and with added P (P1) by 42 d. The effect on growth due to mycorrhizal symbiosis (i.e. mycorrhizal growth response, % MGR) at 42 d was higher in plants grown in P0 soil. Plants colonized by Glomus sp. ‘City Beach’ had a greater % MGR than plants colonized by S. calospora. Both fungi colonized plants to high levels. The percentage of root length colonized was higher in P0 soil than in P1 soil at 21 d. The internal development of S. calospora appeared less affected by addition of P than Glomus sp. ‘City Beach’ at the early harvests. Formation of arbuscules followed the same trends as total colonization. Shoot P concentration was significantly higher in mycorrhizal plants than in non-mycorrhizal plants, by 21 d in P1 soil and by 28 d in P0 soil. Different percentage responses to added P based on total plant dry weight (% PGR) were observed at 28 and 42 d between the plants colonized by the two fungi. The increased P content due to mycorrhizal colonization (% MPR) differed with soil P. For both fungi grown in their respective soils, the response was greater in plants grown in P0 soil. Although the isolate of S. calospora used is an aggressive arbuscular mycorrhizal fungus with some hosts, it promotes a strong positive plant growth response in A. porrum after a mild initial growth depression.

Individual arbuscular mycorrhizal fungi can differ markedly in their ability to improve the phosphate nutrition and growth of host plants. In particular, Scutellospora calospora is relatively ineffective with some hosts and a growth depression is often observed. We have examined the abilities of S. calospora and Glomus sp. ‘City Beach’ strain WUM 16, grown in soils which promote extensive mycorrhizal colonization, to transfer phosphate (P) to Allium porrum. Phosphate uptake from the low-P soils (P0) was compared with uptake from soils amended with extra P (P1). In order to relate P transfer to physiological characteristics of the two fungi, inflow of P via fungus to the plant was combined with the surface areas of intercellular hyphae and arbuscules (symbiotic interfaces) to calculate the amount of P transferred per unit area of interface (P fluxes). ‘Hyphal inflows’ and ‘hyphal fluxes’ were also calculated on the assumption that P uptake by the plant was the same in mycorrhizal and equivalent non-mycorrhizal (NM) plants (the validity of this assumption is discussed). With both soil P treatments, P was taken up by plants colonized by both mycorrhizal fungi to a greater extent than by the equivalent NM controls. Hyphal inflows to plants in P0 soil that were colonized by Glomus sp. ‘City Beach’ were greatest from 14–21 d and decreased at later harvests. With P1 soil the inflow via Glomus sp. ‘City Beach’ peaked at a very high value at 21–28 d. Hyphal inflows into plants in P0 soil that were colonized by S. calospora increased throughout the experiment, and with P1 soil they remained steady at high values. With both fungi, the surface areas of the symbiotic interfaces increased greatly after 14 d, and generally there was little effect of higher soil P. With P0 soil, Glomus sp. ‘City Beach’ showed no significant differences in the ratio of surface areas of the two interfaces over the course of the experiment. Scutellospora calospora tended to produce a lower percentage of interfacial area contributed by arbuscules. With the mycorrhizal plants growing in their respective soils, there appeared no consistent differences between the two fungi with respect to fluxes of P across the interfaces. With P0 soil, fluxes via Glomus sp. ‘City Beach’ were initially higher than those via S. calospora, but later they were higher with S. calospora. With P1 soil the only difference was the 28–42-d period, when Glomus sp. ‘City Beach’ produced the higher flux (reflecting the low surface area at the time). The results show that relative inefficiency of S. calospora in its ability to transfer P, as reported by others, may result from different environmental conditions, use of different hosts, or even of different fungal isolates (strains).