This Botanical Briefing summarizes current knowledge about myco-heterotrophy, discusses its controversial aspects and highlights future directions for research.

Considerable recent progress has been made in terms of understanding the evolutionary history, germination and nutrition of myco-heterotrophic plants. Myco-heterotrophic plants: (1) are diverse and often ancient lineages that have coevolved with fungi, (2) often demonstrate unusually high specificity towards fungi during germination and maturity, and (3) can either cheat common mycorrhizal networks supported by neighbouring photosynthetic plants to satisfy all or part of their energetic and nutritional needs, or recruit free-living saprotrophic fungi into novel mycorrhizal symbioses. However, several fundamental aspects of myco-heterotrophy remain controversial or unknown, such as symbiotic costs and physiology.

This review addresses the significance of arbuscular mycorrhiza in alleviation of salt stress and their beneficial effects on plant growth and productivity. It also focuses on recent progress in unravelling biochemical, physiological and molecular mechanisms in mycorrhizal plants to alleviate salt stress.

The role of arbuscular mycorrhizal fungi in alleviating salt stress is well documented. This paper reviews the mechanisms arbuscular mycorrhizal fungi employ to enhance the salt tolerance of host plants such as enhanced nutrient acquisition (P, N, Mg and Ca), maintenance of the K⁺ : Na⁺ ratio, biochemical changes (accumulation of proline, betaines, polyamines, carbohydrates and antioxidants), physiological changes (photosynthetic efficiency, relative permeability, water status, abscissic acid accumulation, nodulation and nitrogen fixation), molecular changes (the expression of genes: PIP, Na⁺/H⁺ antiporters, Lsnced, Lslea and LsP5CS) and ultra-structural changes. Theis review identifies certain lesser explored areas such as molecular and ultra-structural changes where further research is needed for better understanding of symbiosis with reference to salt stress for optimum usage of this technology in the field on a large scale. This review paper gives useful benchmark information for the development and prioritization of future research programmes.

In Brunei (Borneo) the expression of the waxy zone throughout plant ontogeny was studied in two taxa of the Nepenthes rafflesiana complex, typica and elongata, which differ in pitcher shape and size. We also tested the adaptive significance of this zone by comparing the trapping efficiency and the number of prey captured of wax-bearing and wax-lacking plants.

In elongata, the waxy zone is always well expanded and the elongated pitchers change little in form during plant development. Wax efficiently traps experimental ants but the number of captured prey in pitchers is low. In contrast, in typica, the waxy zone is reduced in successively produced pitchers until it is lost at the end of the plant's juvenile stage. The form of pitchers thus changes continuously throughout plant ontogeny, from elongated to ovoid. In typica, the number of captured prey is greater, but the role of wax in trapping is minor compared with that of the digestive liquid, and waxy plants do not show a higher insect retention and prey abundance as compared with non-waxy plants.

The waxy zone is not always a key trapping structure in Nepenthes and can be lost when supplanted by more efficient features. This study points out how pitcher structure is submitted to selection, and that evolutionary changes in developmental mechanisms could play a role in the morphological diversity of Nepenthes.

The stomatal conductance of leaves on the main stem was measured periodically under favourable field conditions to evaluate g_max, defined as the maximum stomatal conductance at full leaf expansion, for four varieties of soybean and their respective determinate or indeterminate near isogenic lines (NILs). Leaf morphological traits including stomatal density, guard cell length and vein density were also measured.

The value of g_max ranged from 0·383 to 0·754 mol H₂O m^–2 s^–1 across all the genotypes for both years. For the four pairs of varieties, the indeterminate lines exhibited significantly greater g_max, stomatal density, numbers of epidermal cells per unit area and total vein length per unit area than their respective determinate NILs in both years. The guard cell length, leaf mass per area and single leaf size all tended to be greater in the determinate types. The variation of g_max across genotypes and years was well explained by the product of stomatal density and guard cell length (r = 0·86, P < 0·01).

The indeterminate stem growth habit resulted in a greater maximum stomatal conductance for soybean than the determinate habit, and this was attributed to the differences in leaf structure. This raises the further hypothesis that the difference in stem growth habit results in different water use characteristics of soybean plants in the field. Stomatal conductance under favourable conditions can be modified by leaf morphological traits.

Reproductively mature experimental plants were grown from seed collected from wild plants in two of the largest remaining populations. Controlled hand pollinations on potted plants were conducted to determine the level of compatibility of the species and to investigate compatibility within and between populations. Seeds from the hand pollinations were planted in soil, and they were monitored individually, recording time to seed germination (cotyledon emergence). Wild plants were observed in several of the remaining populations to determine which species visited the flowers.

Hand pollination and seed planting experiments indicate that J. reclinata has a mixed mating system: flowers are able to set fruit with viable seeds with self-pollen, but outcross pollen produces significantly greater fruit and seed set than self-pollen (≥50 % for crosses vs. <25 % for self-pollinations). Visitors included a wide array of insect species, primarily of the orders Diptera, Hymenoptera and Lepidoptera. All visitors captured and examined carried J. reclinata pollen, and usually several other types of pollen.

Remnant populations of beach clustervine will have greater reproductive success not only if floral visitor populations are maintained, but also if movement of either pollen or seed takes place between populations. Restoration efforts should include provisions for the establishment and maintenance of pollinator populations.

Molecular diversity was analysed for 736 individuals from 32 populations in the Swiss Alps and adjacent Jura mountains using five polymorphic microsatellite loci. Pollen flow was estimated using pollen grain-sized fluorescent powder. In addition, individual-based Bayesian approaches were applied to examine population structure.

High within-population genetic diversity (H_E = 0·76) and a relatively low inbreeding coefficient (F_IS = 0·022) were found. Genetic differentiation among populations measured with a standardized measure was considerable (G'_ST = 0·53). A significant isolation-by-distance relationship was found (r = 0·62, P < 0·001) and a significant geographic sub-structure, coinciding with proposed postglacial migration patterns. Altitudinal location and size of populations did not influence molecular variation. Direct measures of pollen flow revealed that insect-mediated pollen dispersal was restricted to short distances within a population.

The natural isolation of suitable habitats for C. thyrsoides restricts gene flow among the populations as expected for a monocarpic species with very limited seed dispersal capacities. The observed high within-population genetic diversity in this rare monocarpic perennial is best explained by its outcrossing behaviour, long-lived individuals and overlapping generations. Despite the high within-population genetic diversity, the considerable genetic differentiation and the clear western–eastern differentiation in this species merits consideration in future conservation efforts.

Availability of sugars in planta for stress responses is likely to depend on complex dynamics of soluble sugar accumulation, sucrose–starch partition and organ allocation. The question of potential relationships between endogenous sugar levels and stress responses to atrazine treatment was investigated through analysis of natural genetic accessions of A. thaliana. Parallel quantitative and statistical analysis of biochemical parameters and of stress-sensitive physiological traits was carried out on a set of 11 accessions.

Important natural variation was found between accessions of A. thaliana in pre-stress shoot endogenous sugar levels and responses of plantlets to subsequent atrazine stress. Moreover, consistent trends and statistically significant correlations were detected between specific endogenous sugar parameters, such as the pre-stress end of day sucrose level in shoots, and physiological markers of atrazine tolerance.

These significant relationships between endogenous carbohydrate metabolism and stress response therefore point to an important integration of carbon nutritional status and induction of stress tolerance in plants. The specific correlation between pre-stress sucrose level and greater atrazine tolerance may reflect adaptive mechanisms that link sucrose accumulation, photosynthesis-related stress and sucrose induction of stress defences.

RT-PCR, northern blotting and in situ hybridization were used to analyse the expression pattern of BcMF9, and antisense RNA technology was applied to study the function of this gene.

BcMF9 is expressed in particular in the tapetum and microspore during the late stages of pollen development. Antisense RNA transgenic plants that displayed decreased expression of BcMF9 showed pollen morphological defects that resulted in reduced pollen germination efficiency. Transmission electron microscopy revealed that the homogeneous pectic exintine layer of pollen facing the exterior was over-developed and predominantly occupied the intine, reversing the normal proportional distribution of the internal endintine layer and the external exintine in transgenic pollen. Inhibition of BcMF9 also resulted in break-up of the previously formed tectum and baculae from the beginning of the binucleate stage, as a result of premature degradation of tapetum.

Several lines of evidence, including patterns of BcMF9 expression and phenotypic defects, suggest a sporophytic role in exine patterning, and a gametophytic mode of action of BcMF9 in intine formation. BcMF9 might act as a co-ordinator in the late stages of tapetum degeneration, and subsequently in the regulation of wall material secretion and, in turn, exine formation. BcMF9 might also play a role in intine formation, possibly via regulation of the dynamic metabolism of pectin.

Spores of Colysis decurrens collected in Kagoshima, Japan, were cultured and observed microscopically. Epi-illuminated micrographs of growing gametophytes were captured every 24 h, allowing analysis of the cell lineage of meristems. Light microscopy of resin-sections and scanning electron microscopy were also used.

Contrary to previous assumptions that strap-shaped Colysis gametophytes have no organized meristem, three different types of meristems are formed during development: (1) apical-cell based – responsible for early growth; (2) marginal – further growth, including gametophyte branching; and (3) multicellular – formation of cushions with archegonia. The cushion is two or three layers thick and intermittent. The apical-cell and multicellular meristems are similar to those of cordate gametophytes of other ferns, but the marginal meristem is unique to the strap-shaped gametophyte of this fern.

The strap-shaped gametophytes of C. decurrens may have evolved from ancestors with a cordate shape by insertion of the marginal meristem phase between the first apical-cell-based meristem and subsequent multicellular meristem phases. Repeated retrieval of the marginal meristem at the multicellular meristem phase would result in indefinite prolongation of gametophyte growth, an ecological adaptation to epiphytic habitats.

CW proteins were extracted from hypocotyls of 10- and 18-d-old flax that had been treated or not treated with 0·5 mm Cd(NO₃)₂. PME and PER expression within these extracts was detected by LC/MS, by isoelectric focusing and enzyme activity assays. Transcript expression by RT-PCR of known flax PME and PER genes was also measured in corresponding samples.

In cadmium-treated seedlings, PME activity increased as compared with controls, particularly at day 10. The increased activity of PME was accompanied by increased abundance of both a basic protein isoform (B2) and a particular transcript (Lupme5). In contrast, induction of PER activity by cadmium was highest at day 18. Among the four reported PER genes, Flxper1 and 3 increased in abundance in the presence of cadmium at day 18.

The temporal regulation of Lupme and Flxper genes and of their respective enzyme activities fits the previously reported cadmium-induced structural changes of homogalacturonans within the CWs. After PME-catalysed de-esterification of homogalacturonans, their cross-linking would depend on the activity of PERs interacting with calcium-dimerized blocks and reinforce the cell cohesion during the cadmium-induced swelling.

Callose was localized by aniline blue staining and immunolabelling using an antibody against (1 -> 3)-β-d-glucan. The study was carried out in stomata of untreated material as well as of material treated with: (1) 2-deoxy-d-glucose (2-DDG) or tunicamycin, which inhibit callose synthesis; (2) coumarin or 2,6-dichlorobenzonitrile (dichlobenil), which block cellulose synthesis; (3) cyclopiazonic acid (CPA), which disturbs cytoplasmic Ca²⁺ homeostasis; and (d) cytochalasin B or oryzalin, which disintegrate actin filaments and microtubules, respectively.

In post-cytokinetic stomata significant amounts of callose persisted in the nascent ventral wall. Callose then began degrading from the mid-region of the ventral wall towards its periphery, a process which kept pace with the formation of an ‘internal stomatal pore’ by local separation of the partner plasmalemmata. In differentiating GCs, callose was consistently localized in the developing cell-wall thickenings. In 2-DDG-, tunicamycin- and CPA-affected stomata, callose deposition and internal stomatal pore formation were inhibited. The affected ventral walls and GC wall thickenings contained membranous elements. Stomata recovering from the above treatments formed a stomatal pore by a mechanism different from that in untreated stomata. After coumarin or dichlobenil treatment, callose was retained in the nascent ventral wall for longer than in control stomata, while internal stomatal pore formation was blocked. Actin filament disintegration inhibited internal stomatal pore formation, without any effect on callose deposition.

In A. nidus stomata the time and pattern of callose deposition and degradation play an essential role in internal stomatal pore formation, and callose participates in deposition of the local GC wall thickenings.

Onion epidermal cells were submitted either to an osmotic shock or to a progressive osmotic shift from an osmotic pressure of 2 to 24 MPa to induce plasmolysis. After 30 min in the treatment solution, deplasmolysis was carried out. Cells were observed by microscopy during the whole cycle of dehydration–rehydration.

The application of an osmotic shock to onion cells, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for <1 s, led to the formation of numerous exocytotic and osmocytic vesicles visualized through light and confocal microscopy. In contrast, after application of a progressive osmotic shift, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for 30 min, no vesicles were observed. Additionally, the absence of Hechtian strand connections led to the bursting of vesicles in the case of the osmotic shock.

It is concluded that the kinetics of osmotic dehydration strongly influence vesicle formation in onion cells, and that Hechtian strand connections between protoplasts and exocytotic vesicles are a prerequisite for successful deplasmolysis. These results suggest that a decrease in the area-to-volume ratio of a cell could cause cell death following an osmotic shock.

The requirement for insect pollination was investigated initially by excluding insects from flowers by using mesh bags and comparing immature and mature fruit-set with those of open-pollinated flowers. Those plants that showed a requirement for insect pollination were then tested to compare fruit-set under two additional pollination service scenarios: (1) reduced pollination, with insects excluded from flowers bagged for part of the flowering period, and (2) supplemental pollination, with flowers hand cross-pollinated to test for pollen limitation.

The proportions of flowers setting fruit in blackthorn, hawthorn and ivy were significantly reduced when insects were excluded from flowers by using mesh bags, whereas fruit-set in bramble and dog rose were unaffected. Restricting the exposure of flowers to pollinators had no significant effect on fruit-set. However, blackthorn and hawthorn were found to be pollen-limited, suggesting that the pollination service was inadequate in the study area.

Ensuring strong populations of insect pollinators may be essential to guarantee a winter fruit supply for birds in UK hedgerows.

Field investigations were conducted on pollinator behaviour, flowering phenology and variation in seed set. The effects of self-pollination following controlled self- and cross-pollination were also examined. Selfing rates of differently sized clones were assessed using allozyme markers.

High rates of geitonogamous pollination were associated with large display size. Female fitness at the ramet level decreased with clone size. Fruit and seed set under cross-pollination were significantly higher than those under self-pollination. The pre-dispersal inbreeding depression was estimated as 0·502 based on the difference in seed set per flower between self- and cross-pollinated flowers. Selfing rates of differently sized clones did not differ.

It is concluded that in A. kusnezoffii the negative effects of self-pollination causing reduced female fertility with clone size arise primarily from a strong early-acting inbreeding depression leading to the abortion of selfed embryos prior to seed maturation.

A total of 95 strawberry ramets were sampled from the three sites via transects. Ramets were mapped and genotyped at five variable microsatellite loci. The variability at these five loci was sufficient to assign plants to clones with high confidence, and the spatial pattern of genets was mapped at each site.

A total of 27 distinct multilocus genotypes were identified. Of these, 18 genotypes were detected only once, with the remaining nine detected in multiple ramets. The largest clone was identified in 16 ramets. No genets were shared between sites, and each site exhibited markedly different clonal and sexual recruitment patterns, ranging from two non-overlapping and widespread genets to 19 distinct genets. Only one flowering genet was female; the remainder were hermaphrodites.

Local population history or fine-scale ecological differences can result in dramatically different reproductive patterns at small spatial scales. This finding may be fairly widespread among clonal plant species, and studies that aim to characterize reproductive modes in species capable of asexual reproduction need to evaluate reproductive modes in multiple populations and sites.

Pollen dispersal patterns and male fecundity variation of Shorea leprosula and Shorea parvifolia ssp. parvifolia on Peninsular Malaysian were investigated during two general flowering seasons (2001 and 2002), using a neighbourhood model modified by including terms accounting for variation in male fecundity among individual trees to express heterogeneity in flowering.

The pollen dispersal patterns of the two dipterocarp species were affected by differences in conspecific tree flowering density, and reductions in conspecific tree flowering density led to an increased selfing rate. Active pollen dispersal and a larger number of effective paternal parents were observed for both species in the season of greater magnitude of general flowering (2002).

The magnitude of general flowering, male fecundity variation, and distance between pollen donors and mother trees should be taken into account when attempting to predict the effects of management practices on the self-fertilization and genetic structure of key tree species in tropical forest, and also the sustainability of possible management strategies, especially selective logging regimes.

Experiments were conducted during 30 d of flooding (waterlogging or submergence) with or without shading and subsequent recovery of 20 d under growth conditions. Changes in dry mass, number of branches/tillers, and length of the longest internodes and main stems were recorded to characterize growth responses. Photosynthetic parameters (photosystem II efficiency and non-photochemical quenching) were determined in mature leaves based on chlorophyll a fluorescence measurements.

In both species growth and photosynthesis recovered after the end of the submergence treatment, with recovery of photosynthesis (starting shortly after de-submergence) preceding recovery of growth (pronounced on days 40–50). The effective quantum yield of photosystem II and non-photochemical quenching were diminished during submergence but rapidly increased upon de-submergence. Similar changes were found in all shaded plants, with or without flooding. Submerged plants did not suffer from photoinhibition throughout the recovery period although their growth recovery was retarded.

After sudden de-submergence the C₃ plant A. philoxeroides and the C₄ plant H. altissima were both able to maintain the functionality of the photosynthetic apparatus through rapid acclimation to changing O₂ and light conditions. The ability for photosynthetic acclimation may be essential for adaptation to wetland habitats in which water levels fluctuate.