Hydrogen peroxide is required for light-induced stomatal opening across different plant species.

Stomatal movement is vital for plants to exchange gases and adaption to terrestrial habitats, which is regulated by environmental and phytohormonal signals. Here, we demonstrate that hydrogen peroxide (H2O2) is required for light-induced stomatal opening. H2O2 accumulates specifically in guard cells even when plants are under unstressed conditions. Reducing H2O2 content through chemical treatments or genetic manipulations results in impaired stomatal opening in response to light. This phenomenon is observed across different plant species, including lycopodium, fern, and monocotyledonous wheat. Additionally, we show that H2O2 induces the nuclear localization of KIN10 protein, the catalytic subunit of plant energy sensor SnRK1. The nuclear-localized KIN10 interacts with and phosphorylates the bZIP transcription factor bZIP30, leading to the formation of a heterodimer between bZIP30 and BRASSINAZOLE-RESISTANT1 (BZR1), the master regulator of brassinosteroid signaling. This heterodimer complex activates the expression of amylase, which enables guard cell starch degradation and promotes stomatal opening. Overall, these findings suggest that H2O2 plays a critical role in light-induced stomatal opening across different plant species.

Are stomata in ferns and allies sluggish? Stomatal responses to CO2 , humidity and light and their scaling with size and density.

Fast stomatal reactions enable plants to successfully cope with a constantly changing environment yet there is an ongoing debate on the stomatal regulation mechanisms in basal plant groups. We measured stomatal morphological parameters in 29 fern and allied species from temperate to tropical biomes and two outgroup angiosperm species. Stomatal dynamic responses to environmental drivers were measured in 16 ferns and the two angiosperms using a gas-exchange system. Principal components analyses were used to further reveal the structure-function relationships in stomata. We show a > 10-fold variation for stomatal opening delays and 20-fold variation for stomatal closing delays in ferns. Across species, stomatal responses to vapor pressure deficit (VPD) were the fastest, while light and [CO2 ] responses were slower. In most cases the outgroup species' reaction speeds to changes in environmental variables were similar to those of ferns. Correlations between stomatal response rate and size were apparent for stomatal opening in light and low [CO2 ] while not evident for closing reactions and changes in VPD. No correlations between stomatal density and response speed were observed. Together, this study demonstrates different mechanisms controlling stomatal reactions in ferns at different environmental stimuli, which should be considered in future studies relating stomatal morphology and function.

The sucrose-to-malate ratio correlates with the faster CO2 and light stomatal responses of angiosperms compared to ferns.

Stomatal responses to environmental signals differ substantially between ferns and angiosperms. However, the mechanisms that lead to such different responses remain unclear. Here we investigated the extent to which leaf metabolism contributes to coordinate the differential stomatal behaviour among ferns and angiosperms. Stomata from all species were responsive to light and CO2 transitions. However, fern stomatal responses were slower and minor in both absolute and relative terms. Angiosperms have higher stomatal density, but this is not correlated with speed of stomatal closure. The metabolic responses throughout the diel course and under different CO2 conditions differ substantially among ferns and angiosperms. Higher sucrose content and an increased sucrose-to-malate ratio during high CO2 -induced stomatal closure was observed in angiosperms compared to ferns. Furthermore, the speed of stomatal closure was positively and negatively correlated with sugars and organic acids, respectively, suggesting that the balance between sugars and organic acids aids in explaining the faster stomatal responses of angiosperms. Our results suggest that mesophyll-derived metabolic signals, especially those associated with sucrose and malate, may also be important to modulate the differential stomatal behaviour between ferns and angiosperms, providing important new information that helps in understanding the metabolism-mediated mechanisms regulating stomatal movements across land plant evolution.

Gamma radiation-induced in vitro hormetic apogamy in the fern Pityrogramma calomelanos (L.) link.

Pityrogramma calomelanos (L.) Link, popularly known as "Silver fern" has significant importance as a medicinal plant used traditionally for its astringent, analgesic, anti-haemorrhagic, anti-hypertensive, anti-pyretic and anthelminthic properties. This fern demonstrates an increased morphogenetic potential towards sporophyte formation, upon exposure to low doses of gamma radiation. Young sporophytic leaf crosier cultures were established in vitro on agar based Knop's media with and without 20 g/l sucrose. The cultures were subjected to 60Co radiations in the range of 2.5-100 Gy. Apospory (production of gametophytes on sporophytic tissue without spores) was observed on leaf tissue cultured on Knops media with and without sucrose in P. calomelanos, at the end of 60 days. 5 Gy treated explants showed high number of aposporous gametophytes and was comparable to the control. Other tested doses reduced the aposporous gametophyte production significantly. In the second phase of the experimentation, the cultures were retained on the gametophyte induction media for a period of 4 weeks. Aposporous gametophytes were observed to proliferate with occasional development of antheridia. At the end of 4 weeks, morphogenetic development on the gametophytic tissue resulted in a significantly higher number of apogamous sporophytes (production of sporophytes without fusion of gametes) were obtained on 5 Gy treated tissue as compared to control and all the other treated explants. Apogamous sporophytes thus produced were successfully grown in the greenhouse and transferred to the field. Thus the use of gamma radiation in vitro not only reduced the need for sucrose for induction of apospory in P.calomelanos, it also exhibited hormesis at 5 Gy for improved sporophyte production.

Plastid Phylogenomics Resolve Deep Relationships among Eupolypod II Ferns with Rapid Radiation and Rate Heterogeneity.

The eupolypods II ferns represent a classic case of evolutionary radiation and, simultaneously, exhibit high substitution rate heterogeneity. These factors have been proposed to contribute to the contentious resolutions among clades within this fern group in multilocus phylogenetic studies. We investigated the deep phylogenetic relationships of eupolypod II ferns by sampling all major families and using 40 plastid genomes, or plastomes, of which 33 were newly sequenced with next-generation sequencing technology. We performed model-based analyses to evaluate the diversity of molecular evolutionary rates for these ferns. Our plastome data, with more than 26,000 informative characters, yielded good resolution for deep relationships within eupolypods II and unambiguously clarified the position of Rhachidosoraceae and the monophyly of Athyriaceae. Results of rate heterogeneity analysis revealed approximately 33 significant rate shifts in eupolypod II ferns, with the most heterogeneous rates (both accelerations and decelerations) occurring in two phylogenetically difficult lineages, that is, the Rhachidosoraceae-Aspleniaceae and Athyriaceae clades. These observations support the hypothesis that rate heterogeneity has previously constrained the deep phylogenetic resolution in eupolypods II. According to the plastome data, we propose that 14 chloroplast markers are particularly phylogenetically informative for eupolypods II both at the familial and generic levels. Our study demonstrates the power of a character-rich plastome data set and high-throughput sequencing for resolving the recalcitrant lineages, which have undergone rapid evolutionary radiation and dramatic changes in substitution rates.

Land plants drive photorespiration as higher electron-sink: comparative study of post-illumination transient O2 -uptake rates from liverworts to angiosperms through ferns and gymnosperms.

In higher plants, the electron-sink capacity of photorespiration contributes to alleviation of photoinhibition by dissipating excess energy under conditions when photosynthesis is limited. We addressed the question at which point in the evolution of photosynthetic organisms photorespiration began to function as electron sink and replaced the flavodiiron proteins which catalyze the reduction of O2 at photosystem I in cyanobacteria. Algae do not have a higher activity of photorespiration when CO2 assimilation is limited, and it can therefore not act as an electron sink. Using land plants (liverworts, ferns, gymnosperms, and angiosperms) we compared photorespiration activity and estimated the electron flux driven by photorespiration to evaluate its electron-sink capacity at CO2 -compensation point. In vivo photorespiration activity was estimated by the simultaneous measurement of O2 -exchange rate and chlorophyll fluorescence yield. All C3-plants leaves showed transient O2 -uptake after actinic light illumination (post-illumination transient O2 -uptake), which reflects photorespiration activity. Post-illumination transient O2 -uptake rates increased in the order from liverworts to angiosperms through ferns and gymnosperms. Furthermore, photorespiration-dependent electron flux in photosynthetic linear electron flow was estimated from post-illumination transient O2 -uptake rate and compared with the electron flux in photosynthetic linear electron flow in order to evaluate the electron-sink capacity of photorespiration. The electron-sink capacity at the CO2 -compensation point also increased in the above order. In gymnosperms photorespiration was determined to be the main electron-sink. C3-C4 intermediate species of Flaveria plants showed photorespiration activity, which intermediate between that of C3- and C4-flaveria species. These results indicate that in the first land plants, liverworts, photorespiration started to function as electron sink. According to our hypothesis, the dramatic increase in partial pressure of O2 in the atmosphere about 0.4 billion years ago made it possible to drive photorespiration with higher activity in liverworts.

Ferns, mosses and liverworts as model systems for light-mediated chloroplast movements.

Light-induced chloroplast movement is found in most plant species, including algae and land plants. In land plants with multiple small chloroplasts, under weak light conditions, the chloroplasts move towards the light and accumulate on the periclinal cell walls to efficiently perceive light for photosynthesis (the accumulation response). Under strong light conditions, chloroplasts escape from light to avoid photodamage (the avoidance response). In most plant species, blue light induces chloroplast movement, and phototropin receptor kinases are the blue light receptors. Molecular mechanisms for photoreceptors, signal transduction and chloroplast motility systems are being studied using the model plant Arabidopsis thaliana. However, to further understand the molecular mechanisms and evolutionary history of chloroplast movement in green plants, analyses using other plant systems are required. Here, we review recent works on chloroplast movement in green algae, liverwort, mosses and ferns that provide new insights on chloroplast movement.

A site for sori: Ecophysiology of fertile-sterile leaf dimorphy in ferns.

PREMISE OF THE STUDY: Reproduction often requires significant investment and can move resources away from growth and maintenance; maintaining a balance between reproduction and growth can involve trade-offs. Extreme functional specialization has separated reproduction and photosynthesis in most seed plants, yet ferns use the laminar surface of their fronds for both reproduction and photosynthesis. This dual function selects for a variety of frond morphologies that range from no specialization (monomorphy) to extreme dimorphy between fertile and sterile fronds (holodimorphy). Here we examined the ecological and physiological consequences of variation in frond dimorphy in ferns, evaluated reproductive trade-offs across a dimorphy gradient, and speculate on factors controlling the occurrence of holodimorphy. METHODS: Ecophysiological measurements of photosynthetic rate, water potential, hydraulic conductivity, and gross morphological comparisons of frond area and angle were used to evaluate differences between fertile and sterile fronds. We examined three temperate and three tropical fern species that vary in degree of fertile-sterile dimorphy. KEY RESULTS: Holodimorphic species produced fewer fertile fronds, which had significantly higher respiratory rates than in sterile fronds on the same plant or in any frond produced on monomorphic species; hemidimorphic species were frequently intermediate. We found no differences in vulnerability to cavitation between fertile and sterile fronds. In dimorphic species, fertile fronds had higher (less negative) water potential and lower stipe hydraulic conductivity relative than in sterile fronds. CONCLUSIONS: Fertile-sterile dimorphy in ferns appears to come at considerable carbon cost in holodimorohic species. It is possible that the relative costs of this reproductive system are offset by increased spore dispersal, yet such trade-offs require further exploration.

Two Hymenophyllaceae species from contrasting natural environments exhibit a homoiochlorophyllous strategy in response to desiccation stress.

Hymenophyllaceae is a desiccation tolerant family of Pteridophytes which are poikilohydric epiphytes. Their fronds are composed by a single layer of cells and lack true mesophyll cells and stomata. Although they are associated with humid and shady environments, their vertical distribution varies along the trunk of the host plant with some species inhabiting the drier sides with a higher irradiance. The aim of this work was to compare the structure and function of the photosynthetic apparatus during desiccation and rehydration in two species, Hymenophyllum dentatum and Hymenoglossum cruentum, isolated from a contrasting vertical distribution along the trunk of their hosts. Both species were subjected to desiccation and rehydration kinetics to analyze frond phenotypic plasticity, as well as the structure, composition and function of the photosynthetic apparatus. Minimal differences in photosynthetic pigments were observed upon dehydration. Measurements of ϕPSII (effective quantum yield of PSII), ϕNPQ (quantum yield of the regulated energy dissipation of PSII), ϕNO (quantum yield of non-regulated energy dissipation of PSII), and TL (thermoluminescence) indicate that both species convert a functional photochemical apparatus into a structure which exhibits maximum quenching capacity in the dehydrated state with minimal changes in photosynthetic pigments and polypeptide compositions. This dehydration-induced conversion in the photosynthetic apparatus is completely reversible upon rehydration. We conclude that H. dentatum and H. cruentum are homoiochlorophyllous with respect to desiccation stress and exhibited no correlation between inherent desiccation tolerance and the vertical distribution along the host tree trunk.

Different leaf cost-benefit strategies of ferns distributed in contrasting light habitats of sub-tropical forests.

BACKGROUND AND AIMS: Ferns are abundant in sub-tropical forests in southern China, with some species being restricted to shaded understorey of natural forests, while others are widespread in disturbed, open habitats. To explain this distribution pattern, we hypothesize that ferns that occur in disturbed forests (FDF) have a different leaf cost-benefit strategy compared with ferns that occur in natural forests (FNF), with a quicker return on carbon investment in disturbed habitats compared with old-growth forests. METHODS: We chose 16 fern species from contrasting light habitats (eight FDF and eight FNF) and studied leaf functional traits, including leaf life span (LLS), specific leaf area (SLA), leaf nitrogen and phosphorus concentrations (N and P), maximum net photosynthetic rates (A), leaf construction cost (CC) and payback time (PBT), to conduct a leaf cost-benefit analysis for the two fern groups. KEY RESULTS: The two groups, FDF and FNF, did not differ significantly in SLA, leaf N and P, and CC, but FDF had significantly higher A, greater photosynthetic nitrogen- and phosphorus-use efficiencies (PNUE and PPUE), and shorter PBT and LLS compared with FNF. Further, across the 16 fern species, LLS was significantly correlated with A, PNUE, PPUE and PBT, but not with SLA and CC. CONCLUSIONS: Our results demonstrate that leaf cost-benefit analysis contributes to understanding the distribution pattern of ferns in contrasting light habitats of sub-tropical forests: FDF employing a quick-return strategy can pre-empt resources and rapidly grow in the high-resource environment of open habitats; while a slow-return strategy in FNF allows their persistence in the shaded understorey of old-growth forests.

Photosynthetic Light Responses May Explain Vertical Distribution of Hymenophyllaceae Species in a Temperate Rainforest of Southern Chile.

Some epiphytic Hymenophyllaceae are restricted to lower parts of the host (< 60 cm; 10-100 µmol photons m(-2) s(-1)) in a secondary forest of Southern Chile; other species occupy the whole host height (≥ 10 m; max PPFD > 1000 µmol photons m(-2) s(-1)). Our aim was to study the photosynthetic light responses of two Hymenophyllaceae species in relation to their contrasting distribution. We determined light tolerance of Hymenoglossum cruentum and Hymenophyllum dentatum by measuring gas exchange, PSI and PSII light energy partitioning, NPQ components, and pigment contents. H. dentatum showed lower maximum photosynthesis rates (A max) than H. cruentum, but the former species kept its net rates (An) near Amax across a wide light range. In contrast, in the latter one, An declined at PPFDs > 60 µmol photons m(-2) s(-1). H. cruentum, the shadiest plant, showed higher chlorophyll contents than H. dentatum. Differences in energy partitioning at PSI and PSII were consistent with gas exchange results. H. dentatum exhibited a higher light compensation point of the partitioning of absorbed energy between photochemical Y(PSII) and non-photochemical Y(NPQ) processes. Hence, both species allocated energy mainly toward photochemistry instead of heat dissipation at their light saturation points. Above saturation, H. cruentum had higher heat dissipation than H. dentatum. PSI yield (YPSI) remained higher in H. dentatum than H. cruentum in a wider light range. In both species, the main cause of heat dissipation at PSI was a donor side limitation. An early dynamic photo-inhibition of PSII may have caused an over reduction of the Qa+ pool decreasing the efficiency of electron donation to PSI. In H. dentatum, a slight increase in heat dissipation due to acceptor side limitation of PSI was observed above 300 µmol photons m(-2)s(-1). Differences in photosynthetic responses to light suggest that light tolerance and species plasticity could explain their contrasting vertical distribution.

Intramolecular co-action of two independent photosensory modules in the fern phytochrome 3.

Fern phytochrome3/neochrome1 (phy3/neo1) is a chimeric photoreceptor composed of a phytochrome-chromophore binding domain and an almost full-length phototropin. phy3 thus contains two different light-sensing modules; a red/far-red light receptor phytochrome and a blue light receptor phototropin. phy3 induces both red light- and blue light-dependent phototropism in phototropin-deficient Arabidopsis thaliana (phot1 phot2) seedlings. The red-light response is dependent on the phytochrome module of phy3, and the blue-light response is dependent on the phototropin module. We recently showed that both the phototropin-sensing module and the phytochrome-sensing module mediate the blue light-dependent phototropic response. Particularly under low-light conditions, these two light-sensing modules cooperate to induce the blue light-dependent phototropic response. This intramolecular co-action of two independent light-sensing modules in phy3 enhances light sensitivity, and perhaps allowed ferns to adapt to the low-light canopy conditions present in angiosperm forests.

Photosynthesis limitations in three fern species.

Maximum photosynthesis rates in ferns are generally lower than those of seed plants, but little is known about the limiting factors, which are crucial to understand the evolution of photosynthesis in land plants. To address this issue, a gas exchange/chlorophyll fluorescence analysis was performed in three fern species spanning high phylogenetic range within Polypodiopsida (Osmunda regalis, Blechnum gibbum and Nephrolepis exaltata) to determine their maximum net photosynthesis (AN ), stomatal (gs ) and mesophyll (gm ) conductances to CO2 , and the maximum velocity of carboxylation (Vc,max ). The in vitro Rubisco specificity factor (SC /O ) was also determined. All three species had values for SC /O similar to those typical of seed plants, but values of AN , gs , gm and Vc,max were within the lowest range of those observed in seed plants. In addition, gs was unresponsive to light and CO2 , as already described in other fern species. On the contrary, gm varied with changes CO2 . A quantitative photosynthesis limitation analysis suggested that early land plants (ferns) presented not only stomatal limitations-which were less adjustable to the environment-but also restricted gm and Vc,max , resulting in limited maximum photosynthesis rates.

Using combined measurements for comparison of light induction of stomatal conductance, electron transport rate and CO2 fixation in woody and fern species adapted to different light regimes.

We aimed to understand the relation of photosynthetic rate (A) with g(s) and electron transport rate (ETR) in species of great taxonomic range and light adaptation capability during photosynthetic light induction. We studied three woody species (Alnus formosana, Ardisia crenata and Ardisia cornudentata) and four fern species (Pyrrosia lingus, Asplenium antiquum, Diplazium donianum and Archangiopteris somai) with different light adaptation capabilities. Pot-grown materials received 100 and/or 10% sunlight according to their light adaptation capabilities. At least 4 months after light acclimation, CO(2) and H(2)O exchange and chlorophyll fluorescence were measured simultaneously by equipment in the laboratory. In plants adapted or acclimated to low light, dark-adapted leaves exposed to 500 or 2000 µmol m(-2) s(-1) photosynthetic photon flux (PPF) for 30 min showed low gross photosynthetic rate (P(g)) and short time required to reach 90% of maximum P(g) (). At the initiation of illumination, two broad-leaved understory shrubs and the four ferns, especially ferns adapted to heavy shade, showed higher stomatal conductance (g(s)) than pioneer tree species; materials with higher g(s) had short at both 500 and 2000 µmol m(-2) s(-1) PPF. With 500 or 2000 µmol m(-2) s(-1) PPF, the g(s) for the three woody species increased from 2 to 30 min after the start of illumination, but little change in the g(s) of the four ferns. Thus, P(g) and g(s) were not correlated for all material measured at the same PPF and induction time. However, P(g) was positively correlated with ETR, even though CO(2) assimilation may be influenced by stomatal, biochemical and photoinhibitory limitations. In addition, was closely related to time required to reach 90% maximal ETR for all materials and with two levels of PPF combined. Thus, ETR is a good indicator for estimating the light induction of photosynthetic rate of species, across a wide taxonomic range and light adaptation and acclimation capability.

Light and desiccation responses of some Hymenophyllaceae (filmy ferns) from Trinidad, Venezuela and New Zealand: poikilohydry in a light-limited but low evaporation ecological niche.

BACKGROUND AND AIMS: Hymenophyllaceae (filmy ferns) are typically plants of shady, constantly moist habitats. They attain greatest species diversity and biomass in humid tropical montane forests and temperate hyperoceanic climates. This paper presents ecophysiological data bearing on their worldwide ecological niche space and its limits. METHODS: Chlorophyll fluorescence was used to monitor recovery in desiccation experiments, and for measurements of 95 % saturating irradiance [photosynthetic photon flux density (PPFD(95 %))] of photosynthetic electron flow and other parameters, in the New Zealand Hymenophyllum sanguinolentum, and three species each of Hymenophyllum and Trichomanes from forests in Trinidad and Venezuela. KEY RESULTS: Hymenophyllum sanguinolentum was comparable in desiccation tolerance and light responses with the European species. The more common species in the two tropical forests showed PPFD(95 %) >100 µmol m(-2) s(-1), and withstood moderate desiccation (-40 MPa) for several days. The four most shade-adapted species had PPFD(95 %) ≤51 µmol m(-2) s(-1), and were sensitive to even mild and brief desiccation (-22 MPa for 3 d). CONCLUSIONS: Light and desiccation responses of filmy ferns can be seen as an integrated package. At low light and windspeed in humid forests, net radiation and saturation deficit are low, and diffusion resistance high. Water loss is slow and can be supported by modest conduction from the sub-stratum. With higher irradiance, selection pressure for desiccation tolerance increases progressively. With low light and high humidity, the filmy fern pattern of adaptation is probably optimal, and the vascular plant leaf with mesophyll and stomata offers no advantage in light capture, water economy or CO(2) uptake. Trade-offs between light adaptation and desiccation tolerance, and between stem conduction and water absorption through the leaf surface, underlie adaptive radiation and niche differentiation of species within the family. Hymenophyllaceae are a rare example of an evolutionary shift of adaptive strategy from typical vascular plant adaptation to the poikilohydry most typical of bryophytes.

Heterogeneous light supply affects growth and biomass allocation of the understory fern Diplopterygium glaucum at high patch contrast.

Spatial heterogeneity in resource supply is common and responses to heterogeneous resource supply have been extensively documented in clonal angiosperms but not in pteridophytes. To test the hypotheses that clonal integration can modify responses of pteridophytes to heterogeneous resource supply and the integration effect is larger at higher patch contrast, we conducted a field experiment with three homogeneous and two heterogeneous light treatments on the rhizomatous, understory fern Diplopterygium glaucum in an evergreen broad-leaved forest in East China. In homogeneous treatments, all D. glaucum ramets in 1.5 m×1.5 m units were subjected to 10, 40 and 100% natural light, respectively. In the heterogeneous treatment of low patch contrast, ramets in the central 0.5 m×0.5 m plots of the units were subjected to 40% natural light and their interconnected ramets in the surrounding area of the units to 100%; in the heterogeneous treatment of high patch contrast, ramets in the central plots were subjected to 10% natural light and those in the surrounding area to 100%. In the homogeneous treatments, biomass and number of living ramets in the central plots decreased and number of dead ramets increased with decreasing light supply. At low contrast heterogeneous light supply did not affect performance or biomass allocation of D. glaucum in the central plots, but at high contrast it increased lamina biomass and number of living ramets older than annual and modified biomass allocation to lamina and rhizome. Thus, clonal integration can affect responses of understory ferns to heterogeneous light supply and ramets in low light patches can be supported by those in high light. The results also suggest that effects of clonal integration depend on the degree of patch contrast and a significant integration effect may be found only under a relatively high patch contrast.

Callose implication in stomatal opening and closure in the fern Asplenium nidus.

The involvement of callose in the mechanism of stomatal pore opening and closing in the fern Asplenium nidus was investigated by examination of the pattern of callose deposition in open and closed stomata, and by examination of the effects of callose degradation and inhibition or induction of callose synthesis in stomatal movement. Callose was identified with aniline blue staining and a callose antibody and degraded via beta-1,3-D-glucanase. Callose synthesis was inhibited with 2-deoxy-D-glucose and induced by coumarin or dichlobenil. Stomatal pore opening and closing were assessed by estimation of the stomatal pore width. The open stomata entirely lacked callose, while the closed ones displayed distinct radial fibrillar callose arrays in the external periclinal walls. The latter displayed local bending at the region of callose deposition, a deformation that was absent in the open stomata. Both callose degradation and inhibition of callose synthesis reduced the stomatal ability to open in white light and close in darkness. By contrast, callose synthesis induction considerably improved stomatal pore opening and reduced stomatal closure in same conditions. The present data revealed that: during stomatal closure the external periclinal guard cell walls experience a strong mechanical stress, probably triggering callose synthesis; and that callose participates in stomatal movement.

Optics of sunlit water drops on leaves: conditions under which sunburn is possible.

*It is a widespread belief that plants must not be watered in the midday sunshine, because water drops adhering to leaves can cause leaf burn as a result of the intense focused sunlight. The problem of light focusing by water drops on plants has never been thoroughly investigated. *Here, we conducted both computational and experimental studies of this phyto-optical phenomenon in order to clarify the specific environmental conditions under which sunlit water drops can cause leaf burn. *We found that a spheroid drop at solar elevation angle theta approximately 23 degrees, corresponding to early morning or late afternoon, produces a maximum intensity of focused sunlight on the leaf outside the drop's imprint. Our experiments demonstrated that sunlit glass spheres placed on horizontal smooth Acer platanoides (maple) leaves can cause serious leaf burn on sunny summer days. *By contrast, sunlit water drops, ranging from spheroid to flat lens-shaped, on horizontal hairless leaves of Ginkgo biloba and Acer platanoides did not cause burn damage. However, we showed that highly refractive spheroid water drops held 'in focus' by hydrophobic wax hairs on leaves of Salvinia natans (floating fern) can indeed cause sunburn because of the extremely high light intensity in the focal regions, and the loss of water cooling as a result of the lack of intimate contact between drops and the leaf tissue.

Response of growth and antioxidant enzymes in Azolla plants (Azolla pinnata and Azolla filiculoides) exposed to UV-B.

Effect of ultravilolet-B (0.4 Wm(-2)) irradiation on growth, flavonoid content, lipid peroxidation, proline accumulation and activities of superoxide dismutase and peroxidase was comparatively analysed in Azolla pinnata and Azolla filiculoides. Growth measured as increment in dry weight reduced considerably due to all UV-B treatments. However, the reduction was found to be severe in A. filiculoides as compared to A. pinnata. The level of UV-absorbing compound flavonoids increased significantly in A. pinnata plants whereas only a slight increase in the flavonoid content was observed in A. filiculoides. UV-B exposure led to enhanced production of malondialdehyde (MDA) and electrolyte leakage in A. filiculoides than A. pinnata. Proline accumulation also showed a similar trend. Marked differences in the activity of antioxidant enzymes such as superoxide dismutase (SOD) and peroxidase (POD) was noticed in both the plants exposed to UV-B. Our comparative studies indicate A. pinnata to be better tolerant to UV-B as compared with A. filiculoides which appears to be sensitive.